Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: Immunostaining after Recording ATP-elicited Ca 2+ Signals in pancreatic macrophages (PMs). (A). Representative images of pancreatic lobule loaded with Fluo-4AM before (Ai) and after ATP (10 µM) application (Aii), the arrow indicates the position of the ( n = 8). A corresponding Fluo 4 trace from a PM is shown in Aiii . Corresponding immunostaining of this lobule with antibodies F4/80 Alexa Fluo 647 is shown below ( Aiv) . Hoechst 33342 staining of the same area is shown in Av . Arrow points to ear-like shape of PM nucleus. Overlay of antibody and Hoechst 33342 staining is shown in Avi . Scale bar is 10µm. (B). Immunostaining of another area in a pancreatic lobule with monoclonal F4/80 antibodies labeled with Alexa Fluor 647 (Bi) . Staining of nuclei in the same lobule with Hoechst 33342 (Bii) . Overlay of B i with Bii is shown in Biii . Scale bar is 10µm. (C). Representative images of a pancreatic lobule loaded with Fluo-4AM before (Ci) and after ATP (10 µM) application (Cii) , the arrow indicates the position of the PM. Corresponding Fluo 4 trace is shown in Ciii. Immunostaining of the same area with monoclonal CD11b antibody conjugated with Alexa Fluor 647 ( n = 8) is shown in Civ . Overlay of Cii and Civ is shown in Cv . Scale bar is 10µm.

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Immunostaining, Staining, Labeling

Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: IgG-elicited Ca 2+ Spikes in PMs . (A). Single short Ca 2+ spike occurring after application of IgG (0.1–0.25 mg/mL) in a PM from a control pancreatic lobule. This was an infrequent observation (5 out of 29 cells tested) and is most likely not an IgG-elicited Ca 2+ signal as such single spikes have been also observed in 3 out of 15 cells in the absence of IgG stimulation. (B) . Representative trace of IgG (0.1–0.25 mg/mL)-induced Ca 2+ signals in PMs in pancreatic lobules isolated from mice with AP (FAEE-AP model—48 h). Such oscillations were observed in 9 out of 31 cells. Single short spikes have been observed in 4 out of 31 cells. No oscillations were observed in the absence of stimulation with IgG ( n = 14), while single short spikes have been observed in 2 out of 14 cells. (C). Average Ca 2+ spike frequencies in PMs displaying Ca 2+ signals under the conditions indicated. The frequencies in control PMs, both stimulated with IgG (blue bar) and unstimulated (green), as well as in unstimulated PMs from the FAEE-AP model (48 h, orange bar) were much lower than in PMs from the FAEE-AP model stimulated with IgG (red bar, P < 0.007). (D) . Average Ca 2+ spike duration in PMs displaying Ca 2+ signals under the conditions indicated. Although the average spike duration was longer in the PMs from the FAEE-AP mice stimulated with IgG than under the other conditions, the difference was not statistically different ( P > 0.2). (E). Representative images of immunostaining of PMs in lobules using antibodies F4/80 conjugated with Alexa Fluor 647. Lobules were isolated from control and FAEE-AP 3-day mice (72 h in vivo FAEE-AP model). Scale bar is 20µm. (F). Comparison of the average density of PMs in lobules from control and FAEE-AP 2-day and 3-day mice (48 h and 72 h in vivo FAEE-AP model, respectively). Control, 2.36 ± 0.6 SEM, n = 14; FAEE-AP 2 day, 9.56 ± 1.86 SEM, * P < 0.033, n = 16; FAEE-AP 3 days, 15.37 ± 1.51 SEM, * P < 0.038 as compared to FAEE-AP 2-day, n = 35. The difference between control and FAEE-AP 3-day was very highly significant (**** P < 0.0001).

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Control, Isolation, Immunostaining, In Vivo, Comparison

Journal: Molecular Metabolism

Article Title: Loss of Tbk1 kinase activity protects mice from diet-induced metabolic dysfunction

doi: 10.1016/j.molmet.2018.06.007

Figure Lengend Snippet: Tbk1 Δ/ Δ mice have less inflammation relative to Tbk1 +/ + mice on HFD . (A) Representative 20x images and quantification of cd11b/c stained subcutaneous white adipose tissue from Tbk1 Δ/Δ and Tbk1 +/+ mice after 10 weeks on ND or HFD (n = 4–8 mice/group). Scale bar indicates 200 μM and ‘CLS’ refers to crown like structures. (B) mRNA expression of genes encoding CD11c, F4/80, TNFα, IL-6 and IL-12 in subcutaneous WAT of Tbk1 Δ/Δ and Tbk1 +/+ mice fed with HFD as indicated (n = 4–6 mice/group). (C) Liver tissue lysates from 14-week-old HFD-fed Tbk1 Δ/Δ and Tbk1 +/+ mice were immunoblotted with antibodies against IL-6 and IL-1β. β-actin and GAPDH were used as internal loading controls. Indicated cytokines from WAT (D) and liver tissue (E) lysates of HFD-fed Tbk1 +/+ and Tbk1 Δ/Δ mice were measured by Bio-Rad multiplex array (n = 4–8 mice/group for WAT, n = 8–12 mice/group for liver). All mice are from 129S5 background. Results are representative of mean +/− SEM. Statistical analysis by Student's t -test. * p < 0.05, ** p < 0.01.

Article Snippet: Sections for immunohistochemical analysis were blocked with 20% aquablock and incubated with rabbit anti-cd11b/c (Novus Biologicals, NB110-40766) in blocking solution (5% BSA in TBS with 0.05% tween) at 4 °C overnight.

Techniques: Staining, Expressing, Multiplex Assay

Journal: Cell reports

Article Title: BIRC2 Expression Impairs Anti-Cancer Immunity and Immunotherapy Efficacy.

doi: 10.1016/j.celrep.2020.108073

Figure Lengend Snippet: Figure 4. BIRC2 Knockdown in Melanoma Cells Decreases Tumor Growth and Alters Inflammatory Cell Recruitment to the Tumor Micro- environment (A) B16F10 subclones expressing NTC or BIRC2 shRNA (sh3 or sh4) were implanted subcutaneously in female C57BL/6 mice, and tumor growth was monitored. (B–F) Tumors were harvested on day 35 and the percentage of CD8+/CD44+/CD69+ activated T cells (B), CD11b+/NK1.1+ NK cells (C), CD11b+/CD11c+/F4/80

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse BIRC2 Novus Biologicals Cat# NB100-56889 Anti-mouse b-Actin Santa Cruz Biotechnology Cat# sc-47778 Anti-mouse CD3 BioLegend Cat# 102102 Anti-mouse CD3 Novus Biologicals Cat# FAB4841G-100 Anti-mouse CD4 Novus Biologicals Cat# FAB554A-100 Anti-mouse CD8A Novus Biologicals Cat# NBP1-49045PE Anti-mouse CD11b Novus Biologicals Cat# NB110-89474AF405 Anti-mouse CD11c Novus Biologicals Cat# NB110-40766AF488 Anti-mouse CD25 Novus Biologicals Cat# NBP2-27425AF488 Anti-mouse CD28 BioLegend Cat# 100223 Anti-mouse CD44 Novus Biologicals Cat# NBP1-47386APC Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF488 Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF405 Anti-mouse CD69 Novus Biologicals Cat# NBP1-28011AF488 Anti-mouse CD80 Novus Biologicals Cat# NBP1-43385AF488 Anti-mouse CD314 Novus Biologicals Cat# FAB1547V-100UG Anti-mouse F4/80 Novus Biologicals Cat# NB600-404APC Anti-mouse FoxP3 Novus Biologicals Cat# NB100-39002PE Anti-human HIF-1a Novus Biologicals Cat# NB100-479 Anti-human HIF-1b Novus Biologicals Cat# NB100-124 Anti-human HIF-2a Novus Biologicals Cat# NB100-122 Anti-mouse IFNG Novus Biologicals Cat# IC485V-100UG Anti-mouse Ly6c Novus Biologicals Cat# NBP1-28046AF488 Anti-mouse Ly6g Novus Biologicals Cat# FAB1037A-100 Anti-mouse NK1.1 Novus Biologicals Cat# NB100-77528APC Anti-mouse p50 Novus Biologicals Cat# NBP2-6735 Anti-mouse Rel A Novus Biologicals Cat# NB100-2176 Anti-mouse Rel B Novus Biologicals Cat# NBP2-20123 Anti-mouse a-Tubulin Novus Biologicals Cat# NB600-506 Armenian Hamster IgG, anti-mouse CXCL9 (MIG) Bio X Cell Cat# BE0309 Polyclonal Armenian hamster IgG Bio X Cell Cat# BE0091 Syrian Hamster IgG, anti-mouse CTLA-4 Bio X Cell Cat# BP0131 Rat IgG2a, k, anti-mouse PD-1 (CD279) Bio X Cell Cat# BP0146 Rat IgG2a isotype control Bio X Cell Cat# BE0089 Chemicals, Peptides, and Recombinant Proteins Acriflavine Sigma Aldrich SKU # A8126 TRIzol Reagent Invitrogen Cat# 15596026 Puromycin Dihydrochloride ThermoFisher Cat# A1113803 ECL Prime Western Blotting System GE Healthcare SKU# GERPN2232 PolyJet In Vitro DNA Transfection Reagent Signagen Cat # SL100688 Rabbit anti-mouse IgG-HRP Santa Cruz Biotech Cat# sc-358914 Rabbit IgG HRP Linked Whole Ab GE Healthcare SKU# GENA934 (Continued on next page) e1 Cell Reports 32, 108073, August 25, 2020

Techniques: Knockdown, Expressing, shRNA

Journal: Cell reports

Article Title: BIRC2 Expression Impairs Anti-Cancer Immunity and Immunotherapy Efficacy.

doi: 10.1016/j.celrep.2020.108073

Figure Lengend Snippet: Figure 5. BIRC2 Knockdown in Breast Can- cer Cells Decreases Tumor Growth and Al- ters Inflammatory Cell Recruitment to the Tumor Microenvironment (A) EMT6 subclones expressing NTC or either of two shRNAs targeting BIRC2 (sh4 and sh5) were cultured at 20% O2 and analyzed for expression of BIRC2 protein by immunoblot assay. (B) EMT6 subclones (NTC, sh4, and sh5) were im- planted into the mammary fat pad of female BALB/c mice, and tumor volumes were determined (mean ± SEM; n = 4); *p < 0.05 (Kruskal-Wallis test with Benjamini-Hochberg post-test). (C–F) Tumors were harvested on day 13, and the percentage of CD8+/CD44+/CD69+ activated T cells (C), CD3/NK1.1+ NK cells (D), CD11b+/F4/ 80/CD11c+ DCs (E), and CD11b+/Ly6C+ MDSCs (F) was determined (mean ± SEM; n = 4); *p < 0.05 for the indicated pairs (Kruskal-Wallis test with Benjamini-Hochberg post-test). All immune cell populations were calculated as a percentage of the total number of live cells (based on forward and side scatter). (G) EMT6 subclones were implanted into the mammary fat pad of female SCID mice, and tumor growth was monitored. See also Figure S3B.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse BIRC2 Novus Biologicals Cat# NB100-56889 Anti-mouse b-Actin Santa Cruz Biotechnology Cat# sc-47778 Anti-mouse CD3 BioLegend Cat# 102102 Anti-mouse CD3 Novus Biologicals Cat# FAB4841G-100 Anti-mouse CD4 Novus Biologicals Cat# FAB554A-100 Anti-mouse CD8A Novus Biologicals Cat# NBP1-49045PE Anti-mouse CD11b Novus Biologicals Cat# NB110-89474AF405 Anti-mouse CD11c Novus Biologicals Cat# NB110-40766AF488 Anti-mouse CD25 Novus Biologicals Cat# NBP2-27425AF488 Anti-mouse CD28 BioLegend Cat# 100223 Anti-mouse CD44 Novus Biologicals Cat# NBP1-47386APC Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF488 Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF405 Anti-mouse CD69 Novus Biologicals Cat# NBP1-28011AF488 Anti-mouse CD80 Novus Biologicals Cat# NBP1-43385AF488 Anti-mouse CD314 Novus Biologicals Cat# FAB1547V-100UG Anti-mouse F4/80 Novus Biologicals Cat# NB600-404APC Anti-mouse FoxP3 Novus Biologicals Cat# NB100-39002PE Anti-human HIF-1a Novus Biologicals Cat# NB100-479 Anti-human HIF-1b Novus Biologicals Cat# NB100-124 Anti-human HIF-2a Novus Biologicals Cat# NB100-122 Anti-mouse IFNG Novus Biologicals Cat# IC485V-100UG Anti-mouse Ly6c Novus Biologicals Cat# NBP1-28046AF488 Anti-mouse Ly6g Novus Biologicals Cat# FAB1037A-100 Anti-mouse NK1.1 Novus Biologicals Cat# NB100-77528APC Anti-mouse p50 Novus Biologicals Cat# NBP2-6735 Anti-mouse Rel A Novus Biologicals Cat# NB100-2176 Anti-mouse Rel B Novus Biologicals Cat# NBP2-20123 Anti-mouse a-Tubulin Novus Biologicals Cat# NB600-506 Armenian Hamster IgG, anti-mouse CXCL9 (MIG) Bio X Cell Cat# BE0309 Polyclonal Armenian hamster IgG Bio X Cell Cat# BE0091 Syrian Hamster IgG, anti-mouse CTLA-4 Bio X Cell Cat# BP0131 Rat IgG2a, k, anti-mouse PD-1 (CD279) Bio X Cell Cat# BP0146 Rat IgG2a isotype control Bio X Cell Cat# BE0089 Chemicals, Peptides, and Recombinant Proteins Acriflavine Sigma Aldrich SKU # A8126 TRIzol Reagent Invitrogen Cat# 15596026 Puromycin Dihydrochloride ThermoFisher Cat# A1113803 ECL Prime Western Blotting System GE Healthcare SKU# GERPN2232 PolyJet In Vitro DNA Transfection Reagent Signagen Cat # SL100688 Rabbit anti-mouse IgG-HRP Santa Cruz Biotech Cat# sc-358914 Rabbit IgG HRP Linked Whole Ab GE Healthcare SKU# GENA934 (Continued on next page) e1 Cell Reports 32, 108073, August 25, 2020

Techniques: Knockdown, Expressing, Cell Culture, Western Blot

Journal: Cell reports

Article Title: BIRC2 Expression Impairs Anti-Cancer Immunity and Immunotherapy Efficacy.

doi: 10.1016/j.celrep.2020.108073

Figure Lengend Snippet: Figure 6. BIRC2 Knockdown in B16F10 Cells Increases Anti-tumor Immunity by Increasing CXCL9 Expression (A) NTC and BIRC2-KD subclones were implanted into C57BL/6 mice. When BIRC2-KD tumors became palpable, mice were treated with anti-CXCL9 or IgG every 3 days. Tumor volumes were determined (mean ± SEM; n = 4); *p < 0.05 (Kruskal-Wallis test with Benjamini-Hochberg post-test). (B–E) Tumors were harvested on day 35, and the percentage of CD8+ T cells (relative to CD45+ population) (B), CD8+/CD44+/CD69+ T cells (C), CD3/NK1.1+ NK cells (D), and CD11b+/CD11c+/F4/80 DCs (E) was determined (mean ± SEM; n = 4); *p < 0.05 (Kruskal-Wallis test with Benjamini-Hochberg post-test). All immune cell populations (except B) were calculated as a percentage of the total live cells (based on forward and side scatter). (F–H) The Pearson correlation test was performed to compare CXCL9 mRNA expression with CD8+ T cell score (F), NK cell score (G), and DC score (H), using TCGA data from 481 human melanomas. See also Figures S3C and S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse BIRC2 Novus Biologicals Cat# NB100-56889 Anti-mouse b-Actin Santa Cruz Biotechnology Cat# sc-47778 Anti-mouse CD3 BioLegend Cat# 102102 Anti-mouse CD3 Novus Biologicals Cat# FAB4841G-100 Anti-mouse CD4 Novus Biologicals Cat# FAB554A-100 Anti-mouse CD8A Novus Biologicals Cat# NBP1-49045PE Anti-mouse CD11b Novus Biologicals Cat# NB110-89474AF405 Anti-mouse CD11c Novus Biologicals Cat# NB110-40766AF488 Anti-mouse CD25 Novus Biologicals Cat# NBP2-27425AF488 Anti-mouse CD28 BioLegend Cat# 100223 Anti-mouse CD44 Novus Biologicals Cat# NBP1-47386APC Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF488 Anti-mouse CD45 Novus Biologicals Cat# NB100-77417AF405 Anti-mouse CD69 Novus Biologicals Cat# NBP1-28011AF488 Anti-mouse CD80 Novus Biologicals Cat# NBP1-43385AF488 Anti-mouse CD314 Novus Biologicals Cat# FAB1547V-100UG Anti-mouse F4/80 Novus Biologicals Cat# NB600-404APC Anti-mouse FoxP3 Novus Biologicals Cat# NB100-39002PE Anti-human HIF-1a Novus Biologicals Cat# NB100-479 Anti-human HIF-1b Novus Biologicals Cat# NB100-124 Anti-human HIF-2a Novus Biologicals Cat# NB100-122 Anti-mouse IFNG Novus Biologicals Cat# IC485V-100UG Anti-mouse Ly6c Novus Biologicals Cat# NBP1-28046AF488 Anti-mouse Ly6g Novus Biologicals Cat# FAB1037A-100 Anti-mouse NK1.1 Novus Biologicals Cat# NB100-77528APC Anti-mouse p50 Novus Biologicals Cat# NBP2-6735 Anti-mouse Rel A Novus Biologicals Cat# NB100-2176 Anti-mouse Rel B Novus Biologicals Cat# NBP2-20123 Anti-mouse a-Tubulin Novus Biologicals Cat# NB600-506 Armenian Hamster IgG, anti-mouse CXCL9 (MIG) Bio X Cell Cat# BE0309 Polyclonal Armenian hamster IgG Bio X Cell Cat# BE0091 Syrian Hamster IgG, anti-mouse CTLA-4 Bio X Cell Cat# BP0131 Rat IgG2a, k, anti-mouse PD-1 (CD279) Bio X Cell Cat# BP0146 Rat IgG2a isotype control Bio X Cell Cat# BE0089 Chemicals, Peptides, and Recombinant Proteins Acriflavine Sigma Aldrich SKU # A8126 TRIzol Reagent Invitrogen Cat# 15596026 Puromycin Dihydrochloride ThermoFisher Cat# A1113803 ECL Prime Western Blotting System GE Healthcare SKU# GERPN2232 PolyJet In Vitro DNA Transfection Reagent Signagen Cat # SL100688 Rabbit anti-mouse IgG-HRP Santa Cruz Biotech Cat# sc-358914 Rabbit IgG HRP Linked Whole Ab GE Healthcare SKU# GENA934 (Continued on next page) e1 Cell Reports 32, 108073, August 25, 2020

Techniques: Knockdown, Expressing

Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: Myeloid cells dominate the acute response to ICH and contribute to early damage. (A) Single cells were isolated from the right hemisphere of naïve, sham-operated and collagenase-induced ICH mice 24 h post-surgery and immunophenotyped using flow cytometry. (B) Left: gating strategy used during flow cytometry analysis. Right: cell counts are shown from the following populations: CD45 int CD11b + microglia, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ), CD45 hi CD11b − CD45R + B cells, and CD45 hi CD11b − CD3 + T cells. Data presented as mean+s.e.m., n =5, two independent experiments. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001, **** P <0.0001, determined by one-way ANOVA with Tukey's post-hoc test. (C) Formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (left) and ICH (right) cases were immunostained for the myeloid cell marker CD11b (green) and DAPI (blue), with red blood cell (RBC) autofluorescence shown in red. One representative image from three cases per group shown. Scale bars: 100 µm (50 μm in insets). (D) White blood cells were isolated from whole blood of wild-type (WT) ( n =7) and chemokine receptor 2 ( Ccr2 ) −/− ( n =9) male littermate mice. Top left: CD115 + monocytes and Ly6G + neutrophils as a percentage of all CD45 + immune cells. Bottom left: representative flow plots of the circulating monocyte compartment of each genotype. Right: the amount of Ly6C hi monocytes as a percentage of total circulating monocytes from each genotype. ns, not significant; **** P <0.0001, determined by unpaired two-tailed t -test. (E) Following a 3 day training period on the rotarod assay, WT and Ccr2 −/− littermates were subjected to collagenase-induced ICH and tested on the rotarod again 24 h later. Data presented as net difference in latency to fall from baseline performance, n =7-9, four independent experiments. * P <0.05, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Isolation, Flow Cytometry, Formalin-fixed Paraffin-Embedded, Control, Marker, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: IL-1 produced by mononuclear phagocytes controls acute recruitment of myeloid cells during ICH. (A) Mice were subjected to collagenase-induced ICH and culled at 4 h ( n =5) and 24 h ( n =4) post-ICH. 20 µm coronal brain sections were immunostained for either IL-1α (green, top row) or IL-1β (green, bottom row) together with Iba1 (red) and DAPI (blue). Dashed lines represent haematoma border, arrows define haematomal IL-1β+ cells, and insets are higher-magnification images of respective white boxes. Scale bars: 50 µm. (B) Quantification of IL-1 + cells. (C) 8 µm sections of formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (top) and acute ICH (bottom) cases were immunostained for Iba1 (red), IL-1β (green) and DAPI (blue). Red blood cell (RBC) autofluorescence can be seen in white. One representative image from three patients per group is shown. Scale bars: 100 µm. Insets are higher-magnification images of the areas within the white boxes. (D) Representative flow plot of cells isolated from brains of mice injected with central (10 µg intrastriatal) and peripheral (100 mg kg −1 subcutaneous) IL-1 receptor antagonist (IL-1Ra) ( n =4) or placebo ( n =5) and subjected to ICH, two independent experiments. (E) Cell counts are shown from the following populations: CD45 hi CD11b − lymphocytes, CD45 int CD11b + microglia, CD45 hi CD11b + myeloid cells, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ). Data presented as mean+s.e.m. ns, not significant; * P <0.05, ** P <0.01, *** P <0.001, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Produced, Formalin-fixed Paraffin-Embedded, Control, Isolation, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Macrophage extracellular traps promote maladaptive cardiac remodelling and heart failure via PAD4-dependent mechanisms

doi: 10.64898/2026.03.15.711858

Figure Lengend Snippet: A , Schematic overview of the experimental design. WT-BMT and PAD4KO-BMT mice received either IgG isotype control or anti-Ly6G antibody (100 μg per mouse) by intraperitoneal injection every 3 days for 28 days, as indicated. B , Peripheral blood cell counts, and neutrophil and monocyte fractions (n=4-5). Neutrophils were determined by CD45 high Gr-1 high CD11b low and monocytes were determined by CD45 high Gr-1 low CD11b high by FACS analysis. C , Echocardiographic assessment 28 days after TAC (n=6 in each group). D , Representative fluorescent immunohistochemistry images of left ventricular tissue stained for neutrophil elastase (NE, green), citrullinated histone H3 (CitH3, red), and troponin I (yellow), with nuclear counterstaining by DAPI (blue). Arrowheads indicate NET-positive cells (NE + CitH3 + ). Scale bars, 50 μm. E , Quantification of the numbers of neutrophils (NE + ) and NETs (NE + CitH3 + ) per myocardial tissue area (n=4). F , Representative left ventricular sections stained for CD68 (green), CitH3 (red), troponin I (yellow) and DAPI (blue). Arrowheads indicate MET-positive cells (CD68 + CitH3 + ). Scale bars, 50 μm. G , Quantification of CD68 + macrophages and METs (CD68 + CitH3 + ) per tissue area (n=3-4). All data are presented as mean ± SEM. *P < 0.05 versus the corresponding IgG-treated group, † P < 0.05 versus IgG-treated WT-BMT mice, and ‡ P < 0.05 versus anti-Ly6G-treated WT-BMT mice as determined by one-way analysis of variance with Tukey’s post hoc analysis.

Article Snippet: Magnetic-activated cell sorting was performed on bone marrow cells using MACS MS columns (Miltenyi Biotec GmbH) with CD11b MicroBeads (130-097-142, Miltenyi Biotec GmbH) according to the manufacturer’s instructions.

Techniques: Control, Injection, Immunohistochemistry, Staining

Journal: The Journal of Clinical Investigation

Article Title: Brain microglia serve as a persistent HIV reservoir despite durable antiretroviral therapy

doi: 10.1172/JCI167417

Figure Lengend Snippet: ( A ) Brain tissue pieces were collected from the indicated brain regions and dissociated by mechanical disruption and enzymatic digestion. A CNS single-cell suspension was generated after Percoll separation. CD3 + T cells were positively selected and used for a CNS T cell QVOA. BrMCs and MG were isolated from the CD3 – fractions by CD11b + selection and by TMEM119 + selection, respectively. For human brains, CD11b + selection was performed to isolate BrMCs. BrMCs or MG at P0 (collected immediately after isolation) were used for purity and phenotype analysis and for RT-qPCR to measure proviral DNA and cell-associated RNA. BrMCs or MG at P1 were cultured 1–2 weeks ex vivo to allow the cells to recover and attach. P1 cells were used for the LRA study and the QVOA. ( B ) MG isolated from ART-suppressed, SIV-infected rhesus macaques were defined by TMEM119 staining (P1 MG) (scale bar: 100 μm) and ( C ) anti-TMEM119/anti-CD11b flow cytometry (P0 MG). ( D ) NHP MG proliferated ex vivo. ( E ) Total and integrated SIV DNA was detectable in isolated P0 MG ( n = 3). ( F ) SIV RNA was induced in isolated P1 MG 7 days after stimulation by the HDACi SAHA (500 nM), but was poorly induced by the canonical NF-κB agonist PEP005 (12 nM), the noncanonical NF-κB agonist AZD5582 (100 nM), or TNF-α (50 ng/mL). *** P < 0.001 compared with mock treatment, by 1-way ANOVA ( n = 3). ( G ) SIV RNA was recovered from the supernatant of NHP P1 MG cocultured with CEM174 ( n = 3). Data are presented as the mean ± SEM.

Article Snippet: For NHP samples, CD11b MicroBeads, NHP (catalog 130-091-100, Miltenyi Biotec) were used ( ).

Techniques: Disruption, Suspension, Generated, Isolation, Selection, Quantitative RT-PCR, Cell Culture, Ex Vivo, Infection, Staining, Flow Cytometry