Review



anti pnfκb pp65  (Cell Signaling Technology Inc)


Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 99
      Buy from Supplier

    Structured Review

    Cell Signaling Technology Inc anti pnfκb pp65
    Anti Pnfκb Pp65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pp65/Phospho-NF-kappaB+p65+(Ser536)+Rabbit+mAb/pmc12913675-361-33-35
    Average 99 stars, based on 9335 article reviews
    anti pnfκb pp65 - by Bioz Stars, 2026-10
    99/100 stars

    Images

    Related Articles

    other:

    Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5
    Article Snippet: Rabbit-Anti-Phospho-NF-κB p65 (Ser536) , Cell Signaling Technology , Cat# 3033; RRID: AB_331284.

    Incubation:

    Article Title: Hepatocyte RIPK1 scaffolding function protects against alcohol-induced liver injury by restraining ER stress-driven apoptosis and inflammation
    Article Snippet: Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–12% acrylamide gel (36255ES, Yeasen, China) and transferred onto 0.22 μm PVDF membranes (Millipore Corporation, USA). .. Membranes were incubated with primary antibodies [anti-RIPK1 (3493S, CST, USA), anti-phospho-MLKL (S345) (37333S, CST, USA), anti-MLKL (37705S, CST, USA), anti-Cleaved-Caspase-3 (9661S, CST, USA), antiTNF-α (60291-1-Ig, Proteintech, China), anti-phospho-IRE1α (ab48187, Abcam, USA), anti-IRE1α (3294S, CST, USA), anti-GRP78 (3177S, CST, USA), anti-CHOP (2895S, CST, USA), anti-p65 (8242S, CST, USA), antiphospho-p65 (3033S, CST, USA), anti-MYC (AE070, ABclonal, China), anti-PERK (3192S, CST, USA), anti-ATF6 (HA722722, HUABIO, China), anti-ATF4 (A27233, ABclonal, China), anti-phospho-eIF2α (AO0692, ABclonal, China), anti-eIF2α (A21221, ABclonal, China), and anti-GAPDH (GB11002, Servicebio, China)] overnight at 4 °C, washed with TBS-T (0.1% Tween-20), and incubated with suitable secondary antibodies [goat anti-mouse IgG HRP (HA1006, HUABIO, China) or goat anti-rabbit IgG HRP (HA1001, HUABIO, China)] at room temperature for 1 h. A chemiluminescent reagent (P0018AS, Beyotime, China) and a CLiNX ChemiDoc MP Imaging System (China) were used for detection. .. For densitometric analysis, band intensities were quantified using ImageJ software.

    Article Title: Inhibition of PKM2 by shikonin ameliorates ferroptosis in acute kidney injury by suppressing the mitochondrial DNA-triggered cGAS-STING pathway
    Article Snippet: After homogenization of kidney tissue or cells in RIPA buffer (P0013B; Beyotime), samples were centrifuged at 12,000 g for 15 min at 4°C, and the supernatants were used for SDS-PAGE. .. Following transfer onto PVDF membranes (Millipore), the membranes were blocked using 5% nonfat milk and incubated with the indicated primary antibodies, including anti-PKM2 (15822- 1-AP; Proteintech; 1:1000), anti-cGAS (31659; Cell Signaling Technology (CST); 1:1000), antiphospho-STING (72971; CST; 1:1000), anti-phospho-TBK1 (ab109272; Abcam; 1:1000), antiTBK1 (38066; CST; 1:1000), anti-TNFα (60291-1-AP; Proteintech; 1:4000), anti-IL-6 (21865-1- AP; Proteintech; 1:1000), anti-GPX4 (67763-1-AP; Proteintech; 1:1000), anti-ACSL4 (22401- 1-AP; Proteintech; 1:1000), anti-NF-κB p65 (8242; CST; 1:1000), anti-phospho-NF-κB p65 (Ser536) (3033; CST; 1:1000), anti-NGAL (30700-1-AP; Proteintech), or anti--Actin (66009-l- lg; Proteintech; 1:5000). .. An enhanced chemiluminescence kit (AmerSham Biosciences) was used to develop protein signals and protein levels were quantified using the ImageJ software (72).

    Transcranial Magnetic Stimulation:

    Article Title: Hepatocyte RIPK1 scaffolding function protects against alcohol-induced liver injury by restraining ER stress-driven apoptosis and inflammation
    Article Snippet: Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–12% acrylamide gel (36255ES, Yeasen, China) and transferred onto 0.22 μm PVDF membranes (Millipore Corporation, USA). .. Membranes were incubated with primary antibodies [anti-RIPK1 (3493S, CST, USA), anti-phospho-MLKL (S345) (37333S, CST, USA), anti-MLKL (37705S, CST, USA), anti-Cleaved-Caspase-3 (9661S, CST, USA), antiTNF-α (60291-1-Ig, Proteintech, China), anti-phospho-IRE1α (ab48187, Abcam, USA), anti-IRE1α (3294S, CST, USA), anti-GRP78 (3177S, CST, USA), anti-CHOP (2895S, CST, USA), anti-p65 (8242S, CST, USA), antiphospho-p65 (3033S, CST, USA), anti-MYC (AE070, ABclonal, China), anti-PERK (3192S, CST, USA), anti-ATF6 (HA722722, HUABIO, China), anti-ATF4 (A27233, ABclonal, China), anti-phospho-eIF2α (AO0692, ABclonal, China), anti-eIF2α (A21221, ABclonal, China), and anti-GAPDH (GB11002, Servicebio, China)] overnight at 4 °C, washed with TBS-T (0.1% Tween-20), and incubated with suitable secondary antibodies [goat anti-mouse IgG HRP (HA1006, HUABIO, China) or goat anti-rabbit IgG HRP (HA1001, HUABIO, China)] at room temperature for 1 h. A chemiluminescent reagent (P0018AS, Beyotime, China) and a CLiNX ChemiDoc MP Imaging System (China) were used for detection. .. For densitometric analysis, band intensities were quantified using ImageJ software.

    Imaging:

    Article Title: Hepatocyte RIPK1 scaffolding function protects against alcohol-induced liver injury by restraining ER stress-driven apoptosis and inflammation
    Article Snippet: Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–12% acrylamide gel (36255ES, Yeasen, China) and transferred onto 0.22 μm PVDF membranes (Millipore Corporation, USA). .. Membranes were incubated with primary antibodies [anti-RIPK1 (3493S, CST, USA), anti-phospho-MLKL (S345) (37333S, CST, USA), anti-MLKL (37705S, CST, USA), anti-Cleaved-Caspase-3 (9661S, CST, USA), antiTNF-α (60291-1-Ig, Proteintech, China), anti-phospho-IRE1α (ab48187, Abcam, USA), anti-IRE1α (3294S, CST, USA), anti-GRP78 (3177S, CST, USA), anti-CHOP (2895S, CST, USA), anti-p65 (8242S, CST, USA), antiphospho-p65 (3033S, CST, USA), anti-MYC (AE070, ABclonal, China), anti-PERK (3192S, CST, USA), anti-ATF6 (HA722722, HUABIO, China), anti-ATF4 (A27233, ABclonal, China), anti-phospho-eIF2α (AO0692, ABclonal, China), anti-eIF2α (A21221, ABclonal, China), and anti-GAPDH (GB11002, Servicebio, China)] overnight at 4 °C, washed with TBS-T (0.1% Tween-20), and incubated with suitable secondary antibodies [goat anti-mouse IgG HRP (HA1006, HUABIO, China) or goat anti-rabbit IgG HRP (HA1001, HUABIO, China)] at room temperature for 1 h. A chemiluminescent reagent (P0018AS, Beyotime, China) and a CLiNX ChemiDoc MP Imaging System (China) were used for detection. .. For densitometric analysis, band intensities were quantified using ImageJ software.

    Immunohistochemistry:

    Article Title: Duhuo Jisheng Decoction Mitigates Osteochondral Degeneration in Knee Osteoarthritis through Modulation of Complement C3a/C3AR1-IKKβ-NF-κB Signaling
    Article Snippet: GA (purity ≥98% by high performance liquid 165 chromatography (HPLC), HY-N0764, MedChemExpress, South Brunswick Township, 166 NJ, USA) and the IKKβ inhibitor BMS 345541 (S8044, Selleck Chemicals, Houston, 167 TX, USA) were dissolved in dimethyl sulfoxide (DMSO) at 10 mM stock concentration 168 for mechanistic assays. .. Primary antibodies for immunohistochemistry (IHC), 169 immunofluorescence (IF), and Western Blot (WB) included: anti-COL2 (ab34712, 170 Abcam, Cambridge, UK), anti-MMP13 (ab39012, Abcam, Cambridge, UK), anti-171 NOS2 (YM8628, ImmunoWay, San Jose, CA, USA), anti-C-C motif chemokine ligand 172 2 (CCL2) (YM8646, ImmunoWay, San Jose, CA, USA), anti-interleukin-6 (IL-6) 173 (ab208113, Abcam, Cambridge, UK), anti-C3 (ab11887, Abcam, Cambridge, UK), 174 anti-C3AR1 (ab108129, Abcam, Cambridge, UK), anti-IKKβ (#8943, CST, Danvers, 175 MA, USA), anti-phospho-IKKβ (Ser177/181; #2697, CST, Danvers, MA, USA), anti-176 IκBα (#4814, CST, Danvers, MA, USA), anti-phospho-IκBα (Ser32/36; #9246, CST, 177 Danvers, MA, USA), anti-P65 (#8242, CST, Danvers, MA, USA), anti-phospho-P65 178 (Ser536; #3033, CST, Danvers, MA, USA), and anti-glyceraldehyde-3-phosphate 179 dehydrogenase (GAPDH) (60004-1-Ig, Proteintech, Rosemont, IL, USA). .. Horseradish 180 J urn al Pr e-p roo f peroxidase (HRP)-conjugated secondary antibodies (#7074 and #7076, CST, Danvers, 181 MA, USA) and Alexa Fluor 594-conjugated secondary antibody (A11012, Invitrogen, 182 Carlsbad, CA, USA) were used according to assay requirements.

    Immunofluorescence:

    Article Title: Duhuo Jisheng Decoction Mitigates Osteochondral Degeneration in Knee Osteoarthritis through Modulation of Complement C3a/C3AR1-IKKβ-NF-κB Signaling
    Article Snippet: GA (purity ≥98% by high performance liquid 165 chromatography (HPLC), HY-N0764, MedChemExpress, South Brunswick Township, 166 NJ, USA) and the IKKβ inhibitor BMS 345541 (S8044, Selleck Chemicals, Houston, 167 TX, USA) were dissolved in dimethyl sulfoxide (DMSO) at 10 mM stock concentration 168 for mechanistic assays. .. Primary antibodies for immunohistochemistry (IHC), 169 immunofluorescence (IF), and Western Blot (WB) included: anti-COL2 (ab34712, 170 Abcam, Cambridge, UK), anti-MMP13 (ab39012, Abcam, Cambridge, UK), anti-171 NOS2 (YM8628, ImmunoWay, San Jose, CA, USA), anti-C-C motif chemokine ligand 172 2 (CCL2) (YM8646, ImmunoWay, San Jose, CA, USA), anti-interleukin-6 (IL-6) 173 (ab208113, Abcam, Cambridge, UK), anti-C3 (ab11887, Abcam, Cambridge, UK), 174 anti-C3AR1 (ab108129, Abcam, Cambridge, UK), anti-IKKβ (#8943, CST, Danvers, 175 MA, USA), anti-phospho-IKKβ (Ser177/181; #2697, CST, Danvers, MA, USA), anti-176 IκBα (#4814, CST, Danvers, MA, USA), anti-phospho-IκBα (Ser32/36; #9246, CST, 177 Danvers, MA, USA), anti-P65 (#8242, CST, Danvers, MA, USA), anti-phospho-P65 178 (Ser536; #3033, CST, Danvers, MA, USA), and anti-glyceraldehyde-3-phosphate 179 dehydrogenase (GAPDH) (60004-1-Ig, Proteintech, Rosemont, IL, USA). .. Horseradish 180 J urn al Pr e-p roo f peroxidase (HRP)-conjugated secondary antibodies (#7074 and #7076, CST, Danvers, 181 MA, USA) and Alexa Fluor 594-conjugated secondary antibody (A11012, Invitrogen, 182 Carlsbad, CA, USA) were used according to assay requirements.

    Western Blot:

    Article Title: Duhuo Jisheng Decoction Mitigates Osteochondral Degeneration in Knee Osteoarthritis through Modulation of Complement C3a/C3AR1-IKKβ-NF-κB Signaling
    Article Snippet: GA (purity ≥98% by high performance liquid 165 chromatography (HPLC), HY-N0764, MedChemExpress, South Brunswick Township, 166 NJ, USA) and the IKKβ inhibitor BMS 345541 (S8044, Selleck Chemicals, Houston, 167 TX, USA) were dissolved in dimethyl sulfoxide (DMSO) at 10 mM stock concentration 168 for mechanistic assays. .. Primary antibodies for immunohistochemistry (IHC), 169 immunofluorescence (IF), and Western Blot (WB) included: anti-COL2 (ab34712, 170 Abcam, Cambridge, UK), anti-MMP13 (ab39012, Abcam, Cambridge, UK), anti-171 NOS2 (YM8628, ImmunoWay, San Jose, CA, USA), anti-C-C motif chemokine ligand 172 2 (CCL2) (YM8646, ImmunoWay, San Jose, CA, USA), anti-interleukin-6 (IL-6) 173 (ab208113, Abcam, Cambridge, UK), anti-C3 (ab11887, Abcam, Cambridge, UK), 174 anti-C3AR1 (ab108129, Abcam, Cambridge, UK), anti-IKKβ (#8943, CST, Danvers, 175 MA, USA), anti-phospho-IKKβ (Ser177/181; #2697, CST, Danvers, MA, USA), anti-176 IκBα (#4814, CST, Danvers, MA, USA), anti-phospho-IκBα (Ser32/36; #9246, CST, 177 Danvers, MA, USA), anti-P65 (#8242, CST, Danvers, MA, USA), anti-phospho-P65 178 (Ser536; #3033, CST, Danvers, MA, USA), and anti-glyceraldehyde-3-phosphate 179 dehydrogenase (GAPDH) (60004-1-Ig, Proteintech, Rosemont, IL, USA). .. Horseradish 180 J urn al Pr e-p roo f peroxidase (HRP)-conjugated secondary antibodies (#7074 and #7076, CST, Danvers, 181 MA, USA) and Alexa Fluor 594-conjugated secondary antibody (A11012, Invitrogen, 182 Carlsbad, CA, USA) were used according to assay requirements.

    Cell-Signaling:

    Article Title: Inhibition of PKM2 by shikonin ameliorates ferroptosis in acute kidney injury by suppressing the mitochondrial DNA-triggered cGAS-STING pathway
    Article Snippet: After homogenization of kidney tissue or cells in RIPA buffer (P0013B; Beyotime), samples were centrifuged at 12,000 g for 15 min at 4°C, and the supernatants were used for SDS-PAGE. .. Following transfer onto PVDF membranes (Millipore), the membranes were blocked using 5% nonfat milk and incubated with the indicated primary antibodies, including anti-PKM2 (15822- 1-AP; Proteintech; 1:1000), anti-cGAS (31659; Cell Signaling Technology (CST); 1:1000), antiphospho-STING (72971; CST; 1:1000), anti-phospho-TBK1 (ab109272; Abcam; 1:1000), antiTBK1 (38066; CST; 1:1000), anti-TNFα (60291-1-AP; Proteintech; 1:4000), anti-IL-6 (21865-1- AP; Proteintech; 1:1000), anti-GPX4 (67763-1-AP; Proteintech; 1:1000), anti-ACSL4 (22401- 1-AP; Proteintech; 1:1000), anti-NF-κB p65 (8242; CST; 1:1000), anti-phospho-NF-κB p65 (Ser536) (3033; CST; 1:1000), anti-NGAL (30700-1-AP; Proteintech), or anti--Actin (66009-l- lg; Proteintech; 1:5000). .. An enhanced chemiluminescence kit (AmerSham Biosciences) was used to develop protein signals and protein levels were quantified using the ImageJ software (72).

    Article Title: Omaveloxolone functions as a molecular crowbar-like degrader of STING to alleviate aging-related inflammation
    Article Snippet: .. Antibodies used included anti-p-STING (Ser366) (Cell Signaling Technology, CST, 50907), anti-p-STING (Ser365) (CST, 72971), antiSTING (CST, 13647), anti-p-TBK1 (Ser172) (CST, 5483), anti-TBK1 (CST, 3504), anti-p-IRF3 (Ser396) (CST, 29047), anti-IRF3 (CST, 11904), antiNF-κB p-p65 (Ser536) (CST, 3033), anti-NF-κB p65 (CST, 8242), anti-pSTAT3 (Tyr705) (CST, 9145), anti-STAT3 (CST, 12640), anti-NRF2 (CST, 12721), anti-HA-Tag (CST, 3724), anti-Flag-Tag (CST, 2368), anti-MycTag (CST, 2278), anti-K27-linkage specific ubiquitin (Abcam, ab181537), anti-β-Actin (CST, 4970), anti-GAPDH (CST, 2118), anti-β-Tubulin (Starter, S0B0238), anti-HUWE1 (CST, 5695), anti-TRIM25 (CST, 13773), anti-MID1 (Immunoway, YT2760), anti-RNF219 (Proteintech, 24988-1- a Age (months) 22 23 24 25 10 mg kg-1 OMA (once every two days) 10 mg kg-1 H-151 (daily) Treatment break 10 mg kg-1 OMA (once every two days) 10 mg kg-1 H-151 (daily) End of the study 2.0 1.5 1.0 0.5 0.0 Il6 Tnf Gbp 2 Il1b Ccl 5 Ifi44 Ccl 4 R el at iv e m R N A le ve ls in li v e r 2.5 H-1 51 0 2 4 6 8 H an gi ng ti m e (s ) Ve hic le OM A Ccl 4 Ccl 5 Ifi44 Irf7 Isg1 5 Gbp 2 Cxc l10 Ifit2 3.0 2.0 1.0 0.0R el at iv e m R N A le ve ls in b ra in Br ai n, N eu N DAPI | NeuN | Synaptophysin Yo un g , V eh i c le Ag ed , O M A A g e d , V eh ic le A g ed , H - 1 5 1 Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMA Ki dn ey Li ve r Br ai n, IB A1 Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMAf Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMA p-STING STING p-TBK1 TBK1 ACTB Young Vehicle Aged Vehicle Aged OMA Aged H-151 b c d e h i g Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMA Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMA 2.0 1.5 1.0 0.5 0.0 Isg1 5 Ifitm 3 Cxc l10 Cxc l9 Il1b B2m Ccl 5 Il6 Tnf Irf7 R el at iv e m R N A l e ve ls in k i d ne y kDa -35 -35 -70 -70 -40 P = 0.0003 P < 0.0001 P = 0.0007 P = 0.0005 P = 0.0007 P = 0.0001 P = 0.0010 P = 0.0010 P = 0.0039 P = 0.0032 P = 0.0273 P = 0.0160 P = 0.0139 P = 0.0072 P = 0.0007 P = 0.0008 P < 0.0001 P < 0.0001 P = 0.0003 P = 0.0002 P = 0.0009 P = 0.0004 P = 0.0006 P = 0.0002 P = 0.0107 P = 0.0052 P = 0.0062 P = 0.0002 P = 0.0003 P = 0.0001 P = 0.0022 P = 0.0015 P = 0.0033 P = 0.0013 P = 0.0051 P = 0.0018 P = 0.0010 P = 0.0045 P = 0.0063 P = 0.0027 P = 0.0941 P = 0.0753 P = 0.0832 P = 0.0320 P = 0.1623 P = 0.0444 P = 0.0817 P = 0.0305 P = 0.0584 P = 0.0409 P = 0.0102 P = 0.0038 Nature Communications Article in Press AP), HRP-conjugated anti-rabbit IgG (Promega, W4011), HRPconjugated anti-mouse IgG (Promega, W4021). ..

    Ubiquitin Proteomics:

    Article Title: Omaveloxolone functions as a molecular crowbar-like degrader of STING to alleviate aging-related inflammation
    Article Snippet: .. Antibodies used included anti-p-STING (Ser366) (Cell Signaling Technology, CST, 50907), anti-p-STING (Ser365) (CST, 72971), antiSTING (CST, 13647), anti-p-TBK1 (Ser172) (CST, 5483), anti-TBK1 (CST, 3504), anti-p-IRF3 (Ser396) (CST, 29047), anti-IRF3 (CST, 11904), antiNF-κB p-p65 (Ser536) (CST, 3033), anti-NF-κB p65 (CST, 8242), anti-pSTAT3 (Tyr705) (CST, 9145), anti-STAT3 (CST, 12640), anti-NRF2 (CST, 12721), anti-HA-Tag (CST, 3724), anti-Flag-Tag (CST, 2368), anti-MycTag (CST, 2278), anti-K27-linkage specific ubiquitin (Abcam, ab181537), anti-β-Actin (CST, 4970), anti-GAPDH (CST, 2118), anti-β-Tubulin (Starter, S0B0238), anti-HUWE1 (CST, 5695), anti-TRIM25 (CST, 13773), anti-MID1 (Immunoway, YT2760), anti-RNF219 (Proteintech, 24988-1- a Age (months) 22 23 24 25 10 mg kg-1 OMA (once every two days) 10 mg kg-1 H-151 (daily) Treatment break 10 mg kg-1 OMA (once every two days) 10 mg kg-1 H-151 (daily) End of the study 2.0 1.5 1.0 0.5 0.0 Il6 Tnf Gbp 2 Il1b Ccl 5 Ifi44 Ccl 4 R el at iv e m R N A le ve ls in li v e r 2.5 H-1 51 0 2 4 6 8 H an gi ng ti m e (s ) Ve hic le OM A Ccl 4 Ccl 5 Ifi44 Irf7 Isg1 5 Gbp 2 Cxc l10 Ifit2 3.0 2.0 1.0 0.0R el at iv e m R N A le ve ls in b ra in Br ai n, N eu N DAPI | NeuN | Synaptophysin Yo un g , V eh i c le Ag ed , O M A A g e d , V eh ic le A g ed , H - 1 5 1 Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMA Ki dn ey Li ve r Br ai n, IB A1 Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMAf Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMA p-STING STING p-TBK1 TBK1 ACTB Young Vehicle Aged Vehicle Aged OMA Aged H-151 b c d e h i g Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMA Young, Vehicle Aged, Vehicle Aged, H-151 Aged, OMA 2.0 1.5 1.0 0.5 0.0 Isg1 5 Ifitm 3 Cxc l10 Cxc l9 Il1b B2m Ccl 5 Il6 Tnf Irf7 R el at iv e m R N A l e ve ls in k i d ne y kDa -35 -35 -70 -70 -40 P = 0.0003 P < 0.0001 P = 0.0007 P = 0.0005 P = 0.0007 P = 0.0001 P = 0.0010 P = 0.0010 P = 0.0039 P = 0.0032 P = 0.0273 P = 0.0160 P = 0.0139 P = 0.0072 P = 0.0007 P = 0.0008 P < 0.0001 P < 0.0001 P = 0.0003 P = 0.0002 P = 0.0009 P = 0.0004 P = 0.0006 P = 0.0002 P = 0.0107 P = 0.0052 P = 0.0062 P = 0.0002 P = 0.0003 P = 0.0001 P = 0.0022 P = 0.0015 P = 0.0033 P = 0.0013 P = 0.0051 P = 0.0018 P = 0.0010 P = 0.0045 P = 0.0063 P = 0.0027 P = 0.0941 P = 0.0753 P = 0.0832 P = 0.0320 P = 0.1623 P = 0.0444 P = 0.0817 P = 0.0305 P = 0.0584 P = 0.0409 P = 0.0102 P = 0.0038 Nature Communications Article in Press AP), HRP-conjugated anti-rabbit IgG (Promega, W4011), HRPconjugated anti-mouse IgG (Promega, W4021). ..



    Similar Products

    peptide  (Miltenyi Biotec)
    95
    Miltenyi Biotec peptide
    Peptide, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pp65/PepTivator+CMV+pp65%2C+premium+grade/pmc13664011-102-86-90
    Average 95 stars, based on 1 article reviews
    peptide - by Bioz Stars, 2026-10
    95/100 stars
    		  Buy from Supplier
    peptivator cmv pp65  (Miltenyi Biotec)
    95
    Miltenyi Biotec peptivator cmv pp65
    Activation-induced marker assays are inherently variable and detect heterogeneous subsets of activated CD4 + T cells (A–D) Representative flow cytometry data and quantification of paired raw unstimulated and <t>pp65-stimulated</t> AIM + frequencies among CD4 + T cells (A and B) and CD8 + T cells (C and D). CV (%) is displayed above the responses for each donor. (E and F) Source(s) of variance in CD4 + (E) and CD8 + (F) AIM + cell frequencies calculated after subtracting the corresponding unstimulated AIM + frequencies, using mixed effects analysis, with donor and experiment (technical replicate) as random effects. Graphs show variance components as the percentages of total variance. (G and H) Uniform manifold approximation and projection (UMAP) analysis was performed on CD4 + (G) and CD8 + (H) total AIM + T cells, defined by co-expression of at least one AIM pair. (I and J) FlowSOM clustering of total AIM + CD4 + T cells. (I) Each circle represents one cluster and its relative marker expression; similar clusters are grouped into 8 metaclusters (colored rings); cluster/metacluster spatial proximity indicates similarity. (J) Heatmap showing relative marker expression in the 8 FlowSOM metaclusters. (K) AIM + CD4 + T cell percentages within each FlowSOM metacluster across individual donors. Replicate AIM assays were performed on cryopreserved PBMCs from 5 donors, with 4–8 technical replicates per donor across 2–4 independent experiments; PBMCs were stimulated for 20 h with CMV pp65 peptides. Total AIM + populations from pp65-stimulated samples were pooled from all donors and technical replicates (G–J) or all technical replicates within each donor (K) prior to analysis.
    Peptivator Cmv Pp65, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pp65/PepTivator+CMV+pp65%2C+premium+grade/pmc13390085-407-13-16
    Average 95 stars, based on 1 article reviews
    peptivator cmv pp65 - by Bioz Stars, 2026-10
    95/100 stars
    		  Buy from Supplier
    cmv  (Miltenyi Biotec)
    95
    Miltenyi Biotec cmv
    Activation-induced marker assays are inherently variable and detect heterogeneous subsets of activated CD4 + T cells (A–D) Representative flow cytometry data and quantification of paired raw unstimulated and <t>pp65-stimulated</t> AIM + frequencies among CD4 + T cells (A and B) and CD8 + T cells (C and D). CV (%) is displayed above the responses for each donor. (E and F) Source(s) of variance in CD4 + (E) and CD8 + (F) AIM + cell frequencies calculated after subtracting the corresponding unstimulated AIM + frequencies, using mixed effects analysis, with donor and experiment (technical replicate) as random effects. Graphs show variance components as the percentages of total variance. (G and H) Uniform manifold approximation and projection (UMAP) analysis was performed on CD4 + (G) and CD8 + (H) total AIM + T cells, defined by co-expression of at least one AIM pair. (I and J) FlowSOM clustering of total AIM + CD4 + T cells. (I) Each circle represents one cluster and its relative marker expression; similar clusters are grouped into 8 metaclusters (colored rings); cluster/metacluster spatial proximity indicates similarity. (J) Heatmap showing relative marker expression in the 8 FlowSOM metaclusters. (K) AIM + CD4 + T cell percentages within each FlowSOM metacluster across individual donors. Replicate AIM assays were performed on cryopreserved PBMCs from 5 donors, with 4–8 technical replicates per donor across 2–4 independent experiments; PBMCs were stimulated for 20 h with CMV pp65 peptides. Total AIM + populations from pp65-stimulated samples were pooled from all donors and technical replicates (G–J) or all technical replicates within each donor (K) prior to analysis.
    Cmv, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pp65/PepTivator+CMV+pp65%2C+premium+grade/10__1080_slash_2162402x__2026__2695692-102-21-23
    Average 95 stars, based on 1 article reviews
    cmv - by Bioz Stars, 2026-10
    95/100 stars
    		  Buy from Supplier
    cmv peptivator  (Miltenyi Biotec)
    95
    Miltenyi Biotec cmv peptivator
    Activation-induced marker assays are inherently variable and detect heterogeneous subsets of activated CD4 + T cells (A–D) Representative flow cytometry data and quantification of paired raw unstimulated and <t>pp65-stimulated</t> AIM + frequencies among CD4 + T cells (A and B) and CD8 + T cells (C and D). CV (%) is displayed above the responses for each donor. (E and F) Source(s) of variance in CD4 + (E) and CD8 + (F) AIM + cell frequencies calculated after subtracting the corresponding unstimulated AIM + frequencies, using mixed effects analysis, with donor and experiment (technical replicate) as random effects. Graphs show variance components as the percentages of total variance. (G and H) Uniform manifold approximation and projection (UMAP) analysis was performed on CD4 + (G) and CD8 + (H) total AIM + T cells, defined by co-expression of at least one AIM pair. (I and J) FlowSOM clustering of total AIM + CD4 + T cells. (I) Each circle represents one cluster and its relative marker expression; similar clusters are grouped into 8 metaclusters (colored rings); cluster/metacluster spatial proximity indicates similarity. (J) Heatmap showing relative marker expression in the 8 FlowSOM metaclusters. (K) AIM + CD4 + T cell percentages within each FlowSOM metacluster across individual donors. Replicate AIM assays were performed on cryopreserved PBMCs from 5 donors, with 4–8 technical replicates per donor across 2–4 independent experiments; PBMCs were stimulated for 20 h with CMV pp65 peptides. Total AIM + populations from pp65-stimulated samples were pooled from all donors and technical replicates (G–J) or all technical replicates within each donor (K) prior to analysis.
    Cmv Peptivator, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pp65/PepTivator+CMV+pp65%2C+premium+grade/us12612459-788-10-12
    Average 95 stars, based on 1 article reviews
    cmv peptivator - by Bioz Stars, 2026-10
    95/100 stars
    		  Buy from Supplier
    anti pp65  (Cell Signaling Technology Inc)
    99
    Cell Signaling Technology Inc anti pp65
    Activation-induced marker assays are inherently variable and detect heterogeneous subsets of activated CD4 + T cells (A–D) Representative flow cytometry data and quantification of paired raw unstimulated and <t>pp65-stimulated</t> AIM + frequencies among CD4 + T cells (A and B) and CD8 + T cells (C and D). CV (%) is displayed above the responses for each donor. (E and F) Source(s) of variance in CD4 + (E) and CD8 + (F) AIM + cell frequencies calculated after subtracting the corresponding unstimulated AIM + frequencies, using mixed effects analysis, with donor and experiment (technical replicate) as random effects. Graphs show variance components as the percentages of total variance. (G and H) Uniform manifold approximation and projection (UMAP) analysis was performed on CD4 + (G) and CD8 + (H) total AIM + T cells, defined by co-expression of at least one AIM pair. (I and J) FlowSOM clustering of total AIM + CD4 + T cells. (I) Each circle represents one cluster and its relative marker expression; similar clusters are grouped into 8 metaclusters (colored rings); cluster/metacluster spatial proximity indicates similarity. (J) Heatmap showing relative marker expression in the 8 FlowSOM metaclusters. (K) AIM + CD4 + T cell percentages within each FlowSOM metacluster across individual donors. Replicate AIM assays were performed on cryopreserved PBMCs from 5 donors, with 4–8 technical replicates per donor across 2–4 independent experiments; PBMCs were stimulated for 20 h with CMV pp65 peptides. Total AIM + populations from pp65-stimulated samples were pooled from all donors and technical replicates (G–J) or all technical replicates within each donor (K) prior to analysis.
    Anti Pp65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pp65/Phospho-NF-kappaB+p65+(Ser536)+Rabbit+mAb/pm41865031-405-9-23
    Average 99 stars, based on 1 article reviews
    anti pp65 - by Bioz Stars, 2026-10
    99/100 stars
    		  Buy from Supplier
    pp65 s65  (Cell Signaling Technology Inc)
    86
    Cell Signaling Technology Inc pp65 s65
    Activation-induced marker assays are inherently variable and detect heterogeneous subsets of activated CD4 + T cells (A–D) Representative flow cytometry data and quantification of paired raw unstimulated and <t>pp65-stimulated</t> AIM + frequencies among CD4 + T cells (A and B) and CD8 + T cells (C and D). CV (%) is displayed above the responses for each donor. (E and F) Source(s) of variance in CD4 + (E) and CD8 + (F) AIM + cell frequencies calculated after subtracting the corresponding unstimulated AIM + frequencies, using mixed effects analysis, with donor and experiment (technical replicate) as random effects. Graphs show variance components as the percentages of total variance. (G and H) Uniform manifold approximation and projection (UMAP) analysis was performed on CD4 + (G) and CD8 + (H) total AIM + T cells, defined by co-expression of at least one AIM pair. (I and J) FlowSOM clustering of total AIM + CD4 + T cells. (I) Each circle represents one cluster and its relative marker expression; similar clusters are grouped into 8 metaclusters (colored rings); cluster/metacluster spatial proximity indicates similarity. (J) Heatmap showing relative marker expression in the 8 FlowSOM metaclusters. (K) AIM + CD4 + T cell percentages within each FlowSOM metacluster across individual donors. Replicate AIM assays were performed on cryopreserved PBMCs from 5 donors, with 4–8 technical replicates per donor across 2–4 independent experiments; PBMCs were stimulated for 20 h with CMV pp65 peptides. Total AIM + populations from pp65-stimulated samples were pooled from all donors and technical replicates (G–J) or all technical replicates within each donor (K) prior to analysis.
    Pp65 S65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pp65/pm41813671-842-26-6
    Average 86 stars, based on 1 article reviews
    pp65 s65 - by Bioz Stars, 2026-10
    86/100 stars
    		  Buy from Supplier

    Image Search Results


    Activation-induced marker assays are inherently variable and detect heterogeneous subsets of activated CD4 + T cells (A–D) Representative flow cytometry data and quantification of paired raw unstimulated and pp65-stimulated AIM + frequencies among CD4 + T cells (A and B) and CD8 + T cells (C and D). CV (%) is displayed above the responses for each donor. (E and F) Source(s) of variance in CD4 + (E) and CD8 + (F) AIM + cell frequencies calculated after subtracting the corresponding unstimulated AIM + frequencies, using mixed effects analysis, with donor and experiment (technical replicate) as random effects. Graphs show variance components as the percentages of total variance. (G and H) Uniform manifold approximation and projection (UMAP) analysis was performed on CD4 + (G) and CD8 + (H) total AIM + T cells, defined by co-expression of at least one AIM pair. (I and J) FlowSOM clustering of total AIM + CD4 + T cells. (I) Each circle represents one cluster and its relative marker expression; similar clusters are grouped into 8 metaclusters (colored rings); cluster/metacluster spatial proximity indicates similarity. (J) Heatmap showing relative marker expression in the 8 FlowSOM metaclusters. (K) AIM + CD4 + T cell percentages within each FlowSOM metacluster across individual donors. Replicate AIM assays were performed on cryopreserved PBMCs from 5 donors, with 4–8 technical replicates per donor across 2–4 independent experiments; PBMCs were stimulated for 20 h with CMV pp65 peptides. Total AIM + populations from pp65-stimulated samples were pooled from all donors and technical replicates (G–J) or all technical replicates within each donor (K) prior to analysis.

    Journal: Cell Reports Methods

    Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

    doi: 10.1016/j.crmeth.2026.101458

    Figure Lengend Snippet: Activation-induced marker assays are inherently variable and detect heterogeneous subsets of activated CD4 + T cells (A–D) Representative flow cytometry data and quantification of paired raw unstimulated and pp65-stimulated AIM + frequencies among CD4 + T cells (A and B) and CD8 + T cells (C and D). CV (%) is displayed above the responses for each donor. (E and F) Source(s) of variance in CD4 + (E) and CD8 + (F) AIM + cell frequencies calculated after subtracting the corresponding unstimulated AIM + frequencies, using mixed effects analysis, with donor and experiment (technical replicate) as random effects. Graphs show variance components as the percentages of total variance. (G and H) Uniform manifold approximation and projection (UMAP) analysis was performed on CD4 + (G) and CD8 + (H) total AIM + T cells, defined by co-expression of at least one AIM pair. (I and J) FlowSOM clustering of total AIM + CD4 + T cells. (I) Each circle represents one cluster and its relative marker expression; similar clusters are grouped into 8 metaclusters (colored rings); cluster/metacluster spatial proximity indicates similarity. (J) Heatmap showing relative marker expression in the 8 FlowSOM metaclusters. (K) AIM + CD4 + T cell percentages within each FlowSOM metacluster across individual donors. Replicate AIM assays were performed on cryopreserved PBMCs from 5 donors, with 4–8 technical replicates per donor across 2–4 independent experiments; PBMCs were stimulated for 20 h with CMV pp65 peptides. Total AIM + populations from pp65-stimulated samples were pooled from all donors and technical replicates (G–J) or all technical replicates within each donor (K) prior to analysis.

    Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), or 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

    Techniques: Activation Assay, Marker, Flow Cytometry, Expressing

    Box-Cox transformation-based correction for background enhances signal detection and reduces variability (A) Re-analysis of AIM assay data from SARS-CoV-2 spike peptide-stimulated PBMCs from solid organ transplant recipients ( n = 33) vaccinated with three doses of a COVID-19 mRNA vaccine (PREVENT cohort), or (B) new analysis of CMV pp65-stimulated PBMCs from 6 healthy donors across 3 independent experiments at each of 4 research centers in the CAN-ASC cohort ( n = 52 total). Shown are correlation and linear regression analyses of unstimulated AIM + frequencies with raw antigen-stimulated AIM + frequencies, net responses after subtracting or dividing by the unstimulated condition. CD134 + CD25 + of CD4 + T cells and CD137 + CD69 + of CD8 + T cells are presented. p values are from Spearman’s correlation tests. Solid lines represent linear regression with error bars indicating 95% confidence intervals (CIs). For the CAN-ASC cohort, regression and correlation analyses were performed after correcting for between-donor variations. (C) Box-Cox transformation workflow to correct for AIM + cells in unstimulated controls, using a user-defined parameter λ ϵ[0,1], yielding a stimulation index (SI). (D and E) Three statistical methods to estimate optimal λ values by minimizing correlation between the unstimulated and net stimulated AIM values. Left: linear regression to identify λ giving the highest probability of zero slope ( β ˆ = 0 ), expressed as a posterior probability distribution for λ. Center: Spearman’s correlation to identify the value of λ producing an estimated zero correlation. Right: likelihood profile for λ based on linear regression to estimate the probable optimal value of λ. Values were estimated for (D) PREVENT or (E) CAN-ASC cohorts. (F) ICC values as a function of λ in the CAN-ASC cohort, using the SI for CD4 + and CD8 + AIM responses to CMV pp65. Gray dotted lines show ICC values for raw (untransformed) AIM + frequencies. (G) Comparison of bootstrap-adjusted CVs calculated between technical replicates within donors for raw AIM + frequencies, net subtraction-/division-corrected AIM + frequencies/ratios, or Box-Cox-corrected SIs. Each point is the bootstrap-adjusted CV of technical replicates from one donor. p values represent Dunnett’s multiple comparisons test following one-way repeated-measures ANOVA. (H and I) Re-analysis of AIM responses for the PREVENT cohort throughout a three-dose COVID-19 mRNA vaccination schedule calculated by subtracting or dividing by unstimulated AIM + frequencies, or Box-Cox-corrected SI values. p values represent Dunnett’s multiple comparisons test following mixed-effects analysis of log 2 -transformed data. Bold p values denote significant differences between time points detected only with Box-Cox-corrected SI values.

    Journal: Cell Reports Methods

    Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

    doi: 10.1016/j.crmeth.2026.101458

    Figure Lengend Snippet: Box-Cox transformation-based correction for background enhances signal detection and reduces variability (A) Re-analysis of AIM assay data from SARS-CoV-2 spike peptide-stimulated PBMCs from solid organ transplant recipients ( n = 33) vaccinated with three doses of a COVID-19 mRNA vaccine (PREVENT cohort), or (B) new analysis of CMV pp65-stimulated PBMCs from 6 healthy donors across 3 independent experiments at each of 4 research centers in the CAN-ASC cohort ( n = 52 total). Shown are correlation and linear regression analyses of unstimulated AIM + frequencies with raw antigen-stimulated AIM + frequencies, net responses after subtracting or dividing by the unstimulated condition. CD134 + CD25 + of CD4 + T cells and CD137 + CD69 + of CD8 + T cells are presented. p values are from Spearman’s correlation tests. Solid lines represent linear regression with error bars indicating 95% confidence intervals (CIs). For the CAN-ASC cohort, regression and correlation analyses were performed after correcting for between-donor variations. (C) Box-Cox transformation workflow to correct for AIM + cells in unstimulated controls, using a user-defined parameter λ ϵ[0,1], yielding a stimulation index (SI). (D and E) Three statistical methods to estimate optimal λ values by minimizing correlation between the unstimulated and net stimulated AIM values. Left: linear regression to identify λ giving the highest probability of zero slope ( β ˆ = 0 ), expressed as a posterior probability distribution for λ. Center: Spearman’s correlation to identify the value of λ producing an estimated zero correlation. Right: likelihood profile for λ based on linear regression to estimate the probable optimal value of λ. Values were estimated for (D) PREVENT or (E) CAN-ASC cohorts. (F) ICC values as a function of λ in the CAN-ASC cohort, using the SI for CD4 + and CD8 + AIM responses to CMV pp65. Gray dotted lines show ICC values for raw (untransformed) AIM + frequencies. (G) Comparison of bootstrap-adjusted CVs calculated between technical replicates within donors for raw AIM + frequencies, net subtraction-/division-corrected AIM + frequencies/ratios, or Box-Cox-corrected SIs. Each point is the bootstrap-adjusted CV of technical replicates from one donor. p values represent Dunnett’s multiple comparisons test following one-way repeated-measures ANOVA. (H and I) Re-analysis of AIM responses for the PREVENT cohort throughout a three-dose COVID-19 mRNA vaccination schedule calculated by subtracting or dividing by unstimulated AIM + frequencies, or Box-Cox-corrected SI values. p values represent Dunnett’s multiple comparisons test following mixed-effects analysis of log 2 -transformed data. Bold p values denote significant differences between time points detected only with Box-Cox-corrected SI values.

    Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), or 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

    Techniques: Transformation Assay, Comparison

    AIM responses are reliably detectable across various cell sources (A) Percentage viable of total CD3 + T cells in AIM assays. Error bars represent SD. p values represent comparisons using Dunn’s multiple comparisons test following Friedman test. (B–E) Representative flow cytometry (B and C) and SI quantification (D and E) for each AIM relative to the unstimulated control among CD4 + (B and D) and CD8 + (C and E) T cells from each cell source. (F) Ratio of AIM SIs between CMV pp65 protein- and peptide-stimulated CD4 + T cells in relation to cell source. Positive or negative ratios indicate greater detection of AIM responses with whole protein or peptide stimulation, respectively, while 95% CIs overlapping zero indicate no significant difference between protein and peptide stimulation. AIM responses using fresh whole blood, fresh PBMCs, or cryopreserved PBMCs (PBMC-C), with or without a 24-h delay in processing (−24 h) are compared. Cells were rested overnight and stimulated for 20 h with CMV pp65 peptides, CMV pp65 whole protein, or Infanrix vaccine. Error bars are 95% CIs calculated on a log 2 scale (D, E and F), and data are from n = 6 healthy donors (A–F).

    Journal: Cell Reports Methods

    Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

    doi: 10.1016/j.crmeth.2026.101458

    Figure Lengend Snippet: AIM responses are reliably detectable across various cell sources (A) Percentage viable of total CD3 + T cells in AIM assays. Error bars represent SD. p values represent comparisons using Dunn’s multiple comparisons test following Friedman test. (B–E) Representative flow cytometry (B and C) and SI quantification (D and E) for each AIM relative to the unstimulated control among CD4 + (B and D) and CD8 + (C and E) T cells from each cell source. (F) Ratio of AIM SIs between CMV pp65 protein- and peptide-stimulated CD4 + T cells in relation to cell source. Positive or negative ratios indicate greater detection of AIM responses with whole protein or peptide stimulation, respectively, while 95% CIs overlapping zero indicate no significant difference between protein and peptide stimulation. AIM responses using fresh whole blood, fresh PBMCs, or cryopreserved PBMCs (PBMC-C), with or without a 24-h delay in processing (−24 h) are compared. Cells were rested overnight and stimulated for 20 h with CMV pp65 peptides, CMV pp65 whole protein, or Infanrix vaccine. Error bars are 95% CIs calculated on a log 2 scale (D, E and F), and data are from n = 6 healthy donors (A–F).

    Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), or 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

    Techniques: Flow Cytometry, Control

    A 20-h stimulation period reliably detects most CD4 + and CD8 + T cell AIMs Fresh whole blood or cryopreserved PBMCs (PBMC-C) were stimulated with CMV pp65 peptides for 6, 20, or 44 h. (A) Representative CD134 + CD25 + frequencies of CD4 + T cells. (B) SI quantification of AIM + CD4 + T cells. (C) Representative CD137 + CD69 + frequencies of CD8 + T cells. (D) SI quantification of AIM + CD8 + T cells. p values represent one-sample t tests (B and D), with AIM signals considered present when the mean log 2 -transformed SI significantly differed from zero. Paired samples are from n = 4 healthy donors (A–D).

    Journal: Cell Reports Methods

    Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

    doi: 10.1016/j.crmeth.2026.101458

    Figure Lengend Snippet: A 20-h stimulation period reliably detects most CD4 + and CD8 + T cell AIMs Fresh whole blood or cryopreserved PBMCs (PBMC-C) were stimulated with CMV pp65 peptides for 6, 20, or 44 h. (A) Representative CD134 + CD25 + frequencies of CD4 + T cells. (B) SI quantification of AIM + CD4 + T cells. (C) Representative CD137 + CD69 + frequencies of CD8 + T cells. (D) SI quantification of AIM + CD8 + T cells. p values represent one-sample t tests (B and D), with AIM signals considered present when the mean log 2 -transformed SI significantly differed from zero. Paired samples are from n = 4 healthy donors (A–D).

    Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), or 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

    Techniques: Transformation Assay

    CD134 and CD137 optimally detect antigen-specific Tregs (A) Representative flow cytometric gating strategy for FOXP3 + HELIOS + Tregs. (B) Effect of stimulation with CMV pp65 peptides on FOXP3 + and FOXP3 + HELIOS + frequencies in CD4 + T cells. p values represent Tukey’s multiple comparisons test following two-way ANOVA. (C–E) SI and representative flow cytometry for AIM pairs and Boolean combinations in FOXP3 + HELIOS + Tregs following stimulation of cryopreserved PBMCs with pp65 peptides or whole protein, SARS-CoV-2 spike peptides, or Infanrix combination vaccine for 20 h. Boolean AND/OR gating combinations comprise CD134/CD69, CD134/CD137, and CD137/CD69 (3xAIM); or 3xAIM plus CD154/CD69, CD134/CD154, and CD137/CD154 (6xAIM). Mean log 2 -transformed Box-Cox-corrected SIs are shown. (F) Gating strategy to evaluate CD39 + frequencies among CD134 + CD137 + and non-CD134 + CD137 + FOXP3 + HELIOS + Tregs and FOXP3/HELIOS − Tconvs. (G) Median percentages of CD39 + among CD134 + CD137 + or non-CD134 + CD137 + CD4 + FOXP3 + HELIOS + Tregs or conventional FOXP3/HELIOS − T cells. p values represent Dunnett's multiple comparisons test following repeated measures one-way ANOVA on log2-transformed data. (H) Positive predictive value (PPV) of CD39 or CD39, CD134, and CD137 as markers for antigen-specific Tregs (CD134 + CD137 + FOXP3 + HELIOS + ). Error bars represent 95% confidence intervals of the mean (C, E, and H) or interquartile ranges (G). Healthy donors: n = 9 (pp65 and Infanrix 20-h stimulations) and n = 4 (all others).

    Journal: Cell Reports Methods

    Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

    doi: 10.1016/j.crmeth.2026.101458

    Figure Lengend Snippet: CD134 and CD137 optimally detect antigen-specific Tregs (A) Representative flow cytometric gating strategy for FOXP3 + HELIOS + Tregs. (B) Effect of stimulation with CMV pp65 peptides on FOXP3 + and FOXP3 + HELIOS + frequencies in CD4 + T cells. p values represent Tukey’s multiple comparisons test following two-way ANOVA. (C–E) SI and representative flow cytometry for AIM pairs and Boolean combinations in FOXP3 + HELIOS + Tregs following stimulation of cryopreserved PBMCs with pp65 peptides or whole protein, SARS-CoV-2 spike peptides, or Infanrix combination vaccine for 20 h. Boolean AND/OR gating combinations comprise CD134/CD69, CD134/CD137, and CD137/CD69 (3xAIM); or 3xAIM plus CD154/CD69, CD134/CD154, and CD137/CD154 (6xAIM). Mean log 2 -transformed Box-Cox-corrected SIs are shown. (F) Gating strategy to evaluate CD39 + frequencies among CD134 + CD137 + and non-CD134 + CD137 + FOXP3 + HELIOS + Tregs and FOXP3/HELIOS − Tconvs. (G) Median percentages of CD39 + among CD134 + CD137 + or non-CD134 + CD137 + CD4 + FOXP3 + HELIOS + Tregs or conventional FOXP3/HELIOS − T cells. p values represent Dunnett's multiple comparisons test following repeated measures one-way ANOVA on log2-transformed data. (H) Positive predictive value (PPV) of CD39 or CD39, CD134, and CD137 as markers for antigen-specific Tregs (CD134 + CD137 + FOXP3 + HELIOS + ). Error bars represent 95% confidence intervals of the mean (C, E, and H) or interquartile ranges (G). Healthy donors: n = 9 (pp65 and Infanrix 20-h stimulations) and n = 4 (all others).

    Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), or 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

    Techniques: Flow Cytometry, Transformation Assay

    CMV- and SARS-CoV-2-specific AIM assays are reproducible within and between research centers (A) Experimental design for AIM assay inter-site testing. Replicate aliquots of PBMCs from n = 6 healthy donors assayed in three independent experiments (individual points) by four research groups (coded A–D). Replicate AIM assay experiments ( n = 3 per donor, per site) were performed by stimulating PBMCs for 20 h with CMV pp65 or SARS-CoV-2 spike peptides. (B and C) Box-Cox-corrected SIs of CD134 + CD25 + frequencies among CD4 + T cells and CD137 + CD69 + among CD8 + T cells. Means and SD of log 2 -transformed SIs are shown. Colors indicate the CV within each set of three replicates at each site, with overall CV between sites shown at the top for each donor. (D) Percent CV in CD4 + and CD8 + T cell AIM SIs calculated between technical replicate AIM assays within each site for unstimulated, pp65-stimulated, and spike-stimulated conditions. Each point represents the CV of three technical replicates within one site; bars represent individual donors. CV means with SD are shown. (E and F) ICCs comparing technical variability among CD4 + and CD8 + AIM responses to (E) CMV pp65 and (F) SARS-CoV-2 spike, using data from replicate AIM assays from different sites and donors. Bars represent the mean ICC for each AIM across all sites. (G and H) Percent CV in CD4 + and CD8 + T cell AIM responses to (G) CMV pp65 and (H) SARS-CoV-2 spike between the sites, calculated after averaging three technical replicate AIM assays per site. Each point represents one donor. Error bars represent 95% confidence interval of the mean. (I) ICCs comparing calculated technical variability for replicate AIM assays between sites to biological variability between donors for CD4 + and CD8 + T cells. Dotted lines represent CV = 30% (in D, G and H) and indicate ICC = 0.5 (equal biological and technical variability) (E, F, and I).

    Journal: Cell Reports Methods

    Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

    doi: 10.1016/j.crmeth.2026.101458

    Figure Lengend Snippet: CMV- and SARS-CoV-2-specific AIM assays are reproducible within and between research centers (A) Experimental design for AIM assay inter-site testing. Replicate aliquots of PBMCs from n = 6 healthy donors assayed in three independent experiments (individual points) by four research groups (coded A–D). Replicate AIM assay experiments ( n = 3 per donor, per site) were performed by stimulating PBMCs for 20 h with CMV pp65 or SARS-CoV-2 spike peptides. (B and C) Box-Cox-corrected SIs of CD134 + CD25 + frequencies among CD4 + T cells and CD137 + CD69 + among CD8 + T cells. Means and SD of log 2 -transformed SIs are shown. Colors indicate the CV within each set of three replicates at each site, with overall CV between sites shown at the top for each donor. (D) Percent CV in CD4 + and CD8 + T cell AIM SIs calculated between technical replicate AIM assays within each site for unstimulated, pp65-stimulated, and spike-stimulated conditions. Each point represents the CV of three technical replicates within one site; bars represent individual donors. CV means with SD are shown. (E and F) ICCs comparing technical variability among CD4 + and CD8 + AIM responses to (E) CMV pp65 and (F) SARS-CoV-2 spike, using data from replicate AIM assays from different sites and donors. Bars represent the mean ICC for each AIM across all sites. (G and H) Percent CV in CD4 + and CD8 + T cell AIM responses to (G) CMV pp65 and (H) SARS-CoV-2 spike between the sites, calculated after averaging three technical replicate AIM assays per site. Each point represents one donor. Error bars represent 95% confidence interval of the mean. (I) ICCs comparing calculated technical variability for replicate AIM assays between sites to biological variability between donors for CD4 + and CD8 + T cells. Dotted lines represent CV = 30% (in D, G and H) and indicate ICC = 0.5 (equal biological and technical variability) (E, F, and I).

    Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), or 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

    Techniques: Transformation Assay

    An automated gating pipeline performs comparably to manual analysis for AIM assays (A) Individuals were trained on identical gating protocols and analyzed the same data files from three technical replicates of 20-h AIM assays with CMV pp65 or SARS-CoV-2 spike peptides on healthy donor PBMCs ( n = 6) collected at four sites designated A–D. Raw flow cytometric data from each replicate at each site were analyzed independently by 3 individuals. Data represent CVs between analysts as the mean CV (with error bars showing 95% CIs) for the three technical replicates of each donor-site combination. (B and C) An automated AIM gating pipeline was created; flow cytometric gating approaches comparing manual and automated analyses are shown for (B) CD4 + CD134 + /CD25 + T cells and (C) CD8 + CD137 + /CD69 + T cells. The CytoStim condition was used by the automated software to set donor-specific AIM gates, which were then applied to other stimulations. (D–F) Automated and manual analyses of multi-center CMV and SARS-CoV-2 AIM assay data. A central analyst (C1) defined the gating strategy, provided instructions to the manual analysts (C2 and O1–O4), and was the reference for comparisons. C1, C2, and the automated software each analyzed all data from all sites, while O1–O4 each analyzed data from a single site (O1: A; O2: B; O3: C; O4: D). Box-Cox-corrected AIM SI values are shown for (D) CMV and (E) SARS-CoV-2 AIM assays. p values represent paired comparisons between each analyst and C1 from post-hoc Dunnett’s multiple comparisons test following mixed-effects analysis and were used to compute (F) total numbers of comparisons for C2 and automated analyses that significantly differed from C1 for CD4 + AIMs, with each of the 32 combinations of CD4 + AIM, site, and antigen (CMV or SARS-CoV-2) considered one comparison. Pooled comparisons were analyzed using Fisher’s exact test. (G–I) Spearman’s correlations of AIM SI values for C1, C2, O1–O4 (pooled), and automated analyses for CMV and SARS-CoV-2. Comparisons are shown against (G and H) the reference C1 and (I) for each analyst against all others in a correlation matrix. (J) F1 scores calculated for C2 manual vs. C1 reference (gray) or automated vs. C1 reference (green) analysis. Each point represents the F1 score from a unique donor-site combination, with p values from paired Wilcoxon signed-rank test after averaging F1 scores from technical replicates.

    Journal: Cell Reports Methods

    Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

    doi: 10.1016/j.crmeth.2026.101458

    Figure Lengend Snippet: An automated gating pipeline performs comparably to manual analysis for AIM assays (A) Individuals were trained on identical gating protocols and analyzed the same data files from three technical replicates of 20-h AIM assays with CMV pp65 or SARS-CoV-2 spike peptides on healthy donor PBMCs ( n = 6) collected at four sites designated A–D. Raw flow cytometric data from each replicate at each site were analyzed independently by 3 individuals. Data represent CVs between analysts as the mean CV (with error bars showing 95% CIs) for the three technical replicates of each donor-site combination. (B and C) An automated AIM gating pipeline was created; flow cytometric gating approaches comparing manual and automated analyses are shown for (B) CD4 + CD134 + /CD25 + T cells and (C) CD8 + CD137 + /CD69 + T cells. The CytoStim condition was used by the automated software to set donor-specific AIM gates, which were then applied to other stimulations. (D–F) Automated and manual analyses of multi-center CMV and SARS-CoV-2 AIM assay data. A central analyst (C1) defined the gating strategy, provided instructions to the manual analysts (C2 and O1–O4), and was the reference for comparisons. C1, C2, and the automated software each analyzed all data from all sites, while O1–O4 each analyzed data from a single site (O1: A; O2: B; O3: C; O4: D). Box-Cox-corrected AIM SI values are shown for (D) CMV and (E) SARS-CoV-2 AIM assays. p values represent paired comparisons between each analyst and C1 from post-hoc Dunnett’s multiple comparisons test following mixed-effects analysis and were used to compute (F) total numbers of comparisons for C2 and automated analyses that significantly differed from C1 for CD4 + AIMs, with each of the 32 combinations of CD4 + AIM, site, and antigen (CMV or SARS-CoV-2) considered one comparison. Pooled comparisons were analyzed using Fisher’s exact test. (G–I) Spearman’s correlations of AIM SI values for C1, C2, O1–O4 (pooled), and automated analyses for CMV and SARS-CoV-2. Comparisons are shown against (G and H) the reference C1 and (I) for each analyst against all others in a correlation matrix. (J) F1 scores calculated for C2 manual vs. C1 reference (gray) or automated vs. C1 reference (green) analysis. Each point represents the F1 score from a unique donor-site combination, with p values from paired Wilcoxon signed-rank test after averaging F1 scores from technical replicates.

    Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), or 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

    Techniques: Software, Comparison