Journal: Journal of Biological Chemistry

Article Title: dlk1/FA1 Regulates the Function of Human Bone Marrow Mesenchymal Stem Cells by Modulating Gene Expression of Pro-inflammatory Cytokines and Immune Response-related Factors

doi: 10.1074/jbc.m607530200

Figure Lengend Snippet: FIGURE 5. Dlk1 expression synergizes the effect of LPS-induced cytokines in hMSC cells and stimulates the B cell proliferation. A, control and hMSC-dlk1 cells were stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated and analyzed for the expression of the indicated genes by real time PCR. Expression of each target gene was normalized to -actin. Data are shown as means S.D. of three independent experiments. *, p 0.05; **, p 0.005 versus control cells (hMSC-TERT) induced with LPS. B, CD19-purified human peripheral blood B lymphocytes were cultured in CM obtained from hMSC-TERT (control cells) or hMSC-dlk1 cells as described under “Experimental Procedures” in the absence and the presence of 2.5 or 10 g/ml CD40L. Cell proliferation was measured with XTT-based assay. Stimulation in the cell proliferation was represented as fold induction over nonstimulated cells cultured in the corresponding CM. The bars are the means S.D. of five independent experiments. *, p 0.05; **, p 0.005 versus cultured cells in control/CM.

Article Snippet: Cells were then resuspended at 0.5 106/ml in RPMI (as a positive control), serum-free conditioned medium from hMSC-TERT (control/CM), or hMSC-dlk1 (dlk1/CM) supplemented with 10% FCS and stimulated with 20 ng/ml recombinant human IL-4 (R&D Systems, Abingdon, UK) and the indicated concentrations of recombinant human CD40L (R&D Systems) or left untreated (nonstimulated).

Techniques: Expressing, Control, Isolation, Real-time Polymerase Chain Reaction, Purification, Cell Culture, XTT Assay

Journal: bioRxiv

Article Title: Failed down-regulation of PI3K signaling makes autoreactive B cells receptive to bystander T cell help

doi: 10.1101/2023.01.23.525206

Figure Lengend Snippet: A) Schematic representation of the experimental protocol. One group of OTII x TCRα KO mice was treated with agonistic anti-CD40 antibody on days 6 and 9 post initiation of tamoxifen treatement. B) Proliferation and differentiation to plasmablasts of splenic YFP+ E4+ SHP-1 deficient B cells in WT C57BL/6 mice and OTII x TCRα KO mice, in the presence or absence of agonistic anti-CD40, 14 days after tamoxifen treatment (n=4-5/group, representative cytograms shown). C) Distribution of the YFP+ E4+ SHP-1 deficient Ars/A1 B cell population shown in between an undivided, proliferated and plasmablast (proliferated and CD138+) state. D) Quantification of antibody secreting cells (IgM a anti-Ars) by ELISPOT of spleen cells from . Gray area delineates the limit of detection (50 spots/spleen). E) Quantification of the number of CD45.1+ E4+ cells/spleen in the indicated transfers 14 days after tamoxifen treatment. F) Proliferation and differentiation to plasmablasts of splenic YFP+ E4+ WT Ars/A1 B cells in WT C57BL/6 mice, in the presence or absence of agonistic anti-CD40, 14 days after tamoxifen treatment (n=5/group, representative cytograms shown). G) Distribution of the YFP+ E4+ WT Ars/A1 B cell population shown in between an undivided, proliferated and plasmablast (proliferated and CD138+) state. H) Quantification of antibody secreting cells (IgM a anti-Ars) by ELISPOT of spleen cells from . Gray area delineates the limit of detection (50 spots/spleen). Data shown are representative of at least two replicate experiments. Error bars represent mean ± SEM. Two -tailed unpaired Student’s t test was used. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: In VivoMab anti-mouse CD40, (Clone:FGK4.5/FGK45; Bio X Cell) was injected i.v. or i.p. (200 μl at 0.25mg/ml in sterile PBS) for stimulation.

Techniques: Enzyme-linked Immunospot, Two Tailed Test

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Pembrolizumab does not substantially affect B cell activation and antibody production ex vivo (A-C) Human naïve B cells were isolated from PBMCs and cultured under indicated conditions for 7 days; Pembrolizumab or isotype control IgG4 was added on Day 2, n = 8. (A) Expression of CD38 and CD27 on B cells. Right, percentages of CD27 + CD38 - , CD27 + CD38 + B cells. (B) Expression of CD138 on CD27 + CD38 + B cells. Right, percentage of CD138 + CD27 + CD38 + B cells. (C) Different immunoglobulin isotypes were measured in the culture supernatant of (A-B) by multiplex assay. (D-F) B cells from HuPD-1 mice were isolated and cultured with LPS, IL4, BAFF, or ODN2006, anti-IgM, IL-21, IL-4 or R848, anti-IgM, anti-CD40, IL-21, IFN-γ for 3 days, pembrolizumab or isotype control was added on Day 1. (D) Expression of IgG2c on activated B cells. Right, percentage of IgG2c + B cells, n = 5. (E) Expression of IgG1 on activated B cells. Right, percentage of IgG1 + B cells, n = 5. (F) Different immunoglobulin isotypes in the supernatant of were measured by multiplex assay, n = 5. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: Condition 1: 2.5 μg/mL anti-human Ig (M + G + A) (Jackson Immunoresearch, Cat# 109-006-064), 2.5 μg/mL CpG ODN (Invivogen, Cat# tlrl-2006-1), 10 μg/mL anti-human CD40 (BioXcell, Cat# BE0189), 20 ng/mL rhIL-21 (Peprotech, Cat# 200-21-50UG), 10 ng/mL rhIL-4 (Biolegend, Cat# 574004), 10 ng/mL rhIL-2 (Peprotech, Cat#200-02-250UG).

Techniques: Activation Assay, Ex Vivo, Isolation, Cell Culture, Control, Expressing, Multiplex Assay

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: PD-1 inhibition does not have substantial impact on B cells ex vivo (A) Human naïve B cells were isolated from healthy donor PBMCs and stimulated with different conditions; Pembrolizumab (Keytruda) or isotype control IgG4 was added into the culture media on day 2, mean fluorescence intensity (MFI) of CD86 on B cells was measured, n = 8. (B-F) B cells were isolated from HuPD-1 mice, labeled with CTV, and cultured under 3 different conditions: LPS, rmIL-4, BAFF or Anti-IgM, CpG ODN, rmIL-21, rmIL-4, 100 rhIL-2 or R848, anti-CD40, anti-IgM, rmIL-21, rmIFN-γ for 3 days. Pembrolizumab or isotype control IgG4 was added on day 1, n = 5. (B) Summary of human PD-1 MFI on B cells from different groups. (C) Expression of CD138 on activated B cells. Right, percentage of CD138 + B cells. (D) Summaries of activation marker CD69 and CD86 MFIs on B cells from different groups. (E) Summaries of CD71 and CD98 MFIs on B cells from different groups. (F) Representative flow cytometry plot of CTV dilution on B cells. Right, a summary of the proliferation index of B cells from different groups. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: Condition 1: 2.5 μg/mL anti-human Ig (M + G + A) (Jackson Immunoresearch, Cat# 109-006-064), 2.5 μg/mL CpG ODN (Invivogen, Cat# tlrl-2006-1), 10 μg/mL anti-human CD40 (BioXcell, Cat# BE0189), 20 ng/mL rhIL-21 (Peprotech, Cat# 200-21-50UG), 10 ng/mL rhIL-4 (Biolegend, Cat# 574004), 10 ng/mL rhIL-2 (Peprotech, Cat#200-02-250UG).

Techniques: Inhibition, Ex Vivo, Isolation, Control, Fluorescence, Labeling, Cell Culture, Expressing, Activation Assay, Marker, Flow Cytometry

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Inflammatory signatures enriched in irAE patients reduce antibody production (A-F) Beads based multiplex assays were used to measure plasma concentration of IL-6, and IL-12p70 (A), TNF-α, IFN-γ and IL-1β (B), HC (n = 19), irAE (n = 34), RAC (n = 45), ICI (n = 9). IP-10 (CXCL10), CXCL11, and CXCL9 (C, HC, n = 17; irAE, n = 33; RAC, n = 46; ICI, n = 17), CCL20 (D), CX3CL1 (E), and CCL2 (F). (G-J) Human naïve B cells were isolated and cultured with 0.5 μg/mL anti-human CD40, 2.5 μg/mL anti-human Ig (M+G+A), and 20 ng/mL rhIL-21 with 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, control, or the combination of IFN-α, IL-6, and IL-12 for 7 days. Cells and culture supernatants were analyzed. (G) Representative flow plot of CD38 and CD138 expression on CD27 hi CD38 hi ASCs. Right, a summary of the percentage of CD138 + ASCs, n = 6. (H) Expression of CD11c and CD27 on CD27 - IgD - ASCs. Right, percentage of CD11c + IgD - CD27 - B cells, n = 6. (I) Expression of active-caspase-3 in B cells. Right, percentage of active-caspase-3 + B cells from different groups, n = 3. (J) Different immunoglobulin isotype levels in the culture supernatants from G-H were measured by the multiplex assay, n = 6. Data in graphs represent mean ± SEM, Significance was tested by One-way ANOVA (A-I), and paired Student’s t-test (J).

Article Snippet: Condition 1: 2.5 μg/mL anti-human Ig (M + G + A) (Jackson Immunoresearch, Cat# 109-006-064), 2.5 μg/mL CpG ODN (Invivogen, Cat# tlrl-2006-1), 10 μg/mL anti-human CD40 (BioXcell, Cat# BE0189), 20 ng/mL rhIL-21 (Peprotech, Cat# 200-21-50UG), 10 ng/mL rhIL-4 (Biolegend, Cat# 574004), 10 ng/mL rhIL-2 (Peprotech, Cat#200-02-250UG).

Techniques: Multiplex Assay, Clinical Proteomics, Concentration Assay, Isolation, Cell Culture, Control, Expressing

Journal: Stem Cells

Article Title: Microglia in the spinal cord stem cell niche regulate neural precursor cell proliferation via soluble CD40 in response to myelin basic protein

doi: 10.1093/stmcls/sxae076

Figure Lengend Snippet: Soluble CD40 is the microglia-derived inhibitory factor. (A) Cytokine array analysis reveals 13 factors in the 10-30 kDa molecular weight range that are more highly expressed in MG-spCM with MBP compared to MG - spCM grown in the absence of MBP. The differential intensity was determined by subtracting cytokine intensities of MG-spCM with MBP from MG-spCM minus MBP. ( n = 3 independent experiments) (B) Dose response of mResistin shows inhibition of neurosphere formation at 2000 and 4000 pg/mL ( n = 4 independent experiments, * P < .05, ** P < .01) (C) Dose response of sCD40 shows an inhibitory effect on neurosphere formation at all concentration examined from the spinal cord ( n = 4 independent experiments, **** P < .001). (D) qPCR of CD40 receptor (CD40) and ligand (CD40L) expression in brain and spinal cord derived neurospheres ( n = 3 independent experiments, *** P < .001, * P < .05). (E) Dose response of sCD40 on brain neurosphere formation ( n = 4 independent experiments, **** P < .0001).

Article Snippet: Enzyme-linked immunosorbent assays (ELISAs) were purchased from R&D Systems for CD40 (Catalog #MCCD40) and used for the analysis of microglia-derived CM samples as per the manufacturer's instructions for cell culture supernatant samples.

Techniques: Derivative Assay, Molecular Weight, Inhibition, Concentration Assay, Expressing

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Design of the coiled-coil peptide delivery platform. (A) Anti-CD40 antibody constructs of IgG1 or IgG2 isotypes were connected with E domains of various lengths (EIAALEK)3-6 via a peptide linker (4GS)x2to the C-terminal of the light chain, heavy chain or both light and heavy chains. (B). Combination of antibodies connected with E domains with K domains fused with OVA peptides leads to the formation of complexes. Created with BioRender.com.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: K4-OVA peptides strongly bind E4 domains connected to anti-CD40 antibody constructs and aid formation of stable complexes capable of cell binding and CD40 activation. Binding interactions between E and K coiled coil peptides as determined by ELISA and bio-layer interferometry (BLI). ELISA plates were coated with human CD40 antigen followed by addition of antibody constructs of either IgG1 (A) or IgG2 (B) isotype connected with E4 domains. Biotinylated K4 domains fused with OVA peptides were added followed by detection using streptavidin HRP. Representative BLI affinity measurements to determine binding kinetics of (C) mAbLCE4-IgG1 or (D) mAbLCE4-IgG2 antibody constructs interacting with K4 domains fused with OVA peptides. Antibody constructs connected with E domains were captured on anti-fab 2nd generation (FAB2G) sensors and assayed against serially diluted K4-OVA peptides in solution. Antibody-peptide complex formation and binding of the complexes to B16-F10 CD40 expressing and B16-F10 wt cells was assessed by flow cytometry. IgG1 anti-CD40 antibodies connected with E4 were mixed with equimolar amounts of biotynylated K4-OVA to enable complex formation followed by staining using PE streptavidin and flow cytometry analysis. (E) Cells incubated with 5 µg mAbLCE4 + 0.5 µg biotinylated K4-OVA peptides, (F) cells incubated with 5 µg mAbLCE4, (G) cells incubated with 0.5 µg biotinylated K4-OVA peptides, (H) cells without any compounds. Data shown is representative of two independent experiments. (I) a reporter assay was performed to assess the ability of IgG1 anti-CD40 antibody constructs connected with E domains to induce CD40 activation. CD40 reporter cells (promega) were incubated with FcγRI (CD64) CHO expressing cells or wildtype CHO cells in the presence of mAbLCE3 complexed with K4-OVA peptides, mAbLCE3 and mAb control. CD40 activation was measured according to the manufacturer’s instructions.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct, Binding Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Staining, Incubation, Reporter Assay, Control

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Stability characterization of anti-CD40 antibody constructs connected with E domains. (A) Ability of the constructs to withstand shear stress was determined by measuring A280 before and after rapid agitation. (B) Constructs were incubated in human serum or BSA for seven days after which a Mono ELISA was performed. (C) Stability of the constructs in un optimized buffer (PBS) was analyzed by incubation at room temperature (RT) or 40 °C for 2 weeks followed by SEC-HPLC analysis. Data is presented as changes in high molecular weight species (Δ HMWS) before and after stability studies. (D) Constructs were subjected to three rounds of freeze (-80 °C)-thawing (37 °C) at 24, 48, and 72 hours followed by SEC-HPLC analysis. Data is presented as changes in high molecular weight species (Δ HMWS) before and after stability studies.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct, Shear, Incubation, Enzyme-linked Immunosorbent Assay, High Molecular Weight

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Combining the coiled coil platform with potential antigen carrying nanocarriers. (A) An illustration of formation of antibody-peptide-bead complexes from interactions between antibody-peptide conjugate constructs, biotinylated K6-OVA peptides and streptavidin labeled beads (utilized to mimic antigen loaded nanocarriers). Antibody-peptide-bead complexes were prepared first before addition to cells. (B) B16-F10 CD40 and B16-F10 wt cells incubated with antibody-peptide-bead complexes and stained with both PE anti-streptavidin and anti-human lgG Fc-PE. (C) B16-F10 CD40 and B16-F10 wt cells incubated with antibody-peptide-bead complexes stained with only PE anti-streptavidin. (D) B16-F10 CD40 and B16-F10 wt cells incubated with antibody-peptide-bead complexes stained with only anti-human lgG Fc-PE. (E) Unstained B16-F10 CD40 and B16-F10 wt cells without any compounds. All stainings were followed by FACS analysis. The data depicted here is representative of two independent experiments. was created with BioRender.com.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Construct, Labeling, Incubation, Staining

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Coiled-coil technology enables efficient OVA peptide cross-presentation and activation of OVA-specific CD8+ T cells. (A) Mice treatment plan. On day 0 and day 7, non-tumor bearing male hCD40tg mice were subcutaneously treated with different compound formulations. Seven days after the second treatment (day 14), mice were sacrificed and their inguinal lymph nodes closest to the treatment site (iLN) were removed for FACS analysis of the percentage of OVA peptide-specific CD8+ T cells. (B) Mice were treated with vehicle (PBS), or molar equivalent amounts of the peptides alone or in combination with mAbLCE4 IgG1 construct. (C) a similar treatment set up as that in (B) was used and additional molecules were included (mAbLCE4 -IgG2 + K4-OVA, mAbLCE4 -IgG2+ OVA peptides, mAb wt + OVA peptides). Four to five mice were used per group in each experiment. The graphs show the mean ± SD of data from each experiment. Statisctical analysis was performed using a Mann-Whitney test, * p < 0.05; ** p < 0.01. (D) a postulated mode of action of the technology. After internalization of the antigen-peptide complexes, DCs process and cross-present antigenic peptides to T-cells that are activated to kill tumor cells. (E) An example of a potential application of the technology could be in personalized neoantigen cancer vaccines. From patient tumors, neoantigens are identified and characterized. Neoantigens fused with K domains are manufactured and combined with off-the-shelf anti-CD40 antibodies connected with E domains. Finally, patients are vaccinated with the mixture. were created with BioRender.com.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: Activation Assay, Construct, MANN-WHITNEY, Vaccines, Immunopeptidomics

Journal: Drug Delivery

Article Title: Antigenic peptide delivery to antigen-presenting cells using a CD40-coiled coil affinity-based platform

doi: 10.1080/10717544.2025.2486340

Figure Lengend Snippet: Evaluating the potential of the technology to evoke anti-tumor responses in vivo . (A) Human CD40 transgenic mice were subcutaneously (s.c) administered with (0.2x10 6 ) MB49-EpCam-OVA tumor cells on day 0 followed by s.c. treatment on the right outer flank with either vehicle (PBS), 1.5 µM free OVA-peptides, 0.4 µM of mAbLCE4, 1.5 µM K4-OVA peptides, 0.4 µM of mAbLCE4 in combination with 1.5 µM K4-OVA peptides or 0.4 µg of mAbLCE4 in combination with 1.5 µM OVA peptides on day 10 and 17. (B) Tumor growth and (C) survival over time were monitored. The graphs show the mean (+SEM) of 7–10 mice per group in one experiment. Statistical analysis of tumor volumes was performed on days 21–34 using a Mann-Whitney test. Survival analysis was conducted using Kaplan-Meier log-rank test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns- not statistically significant.

Article Snippet: White, flat-bottom, high-binding 96-well plates (Greiner Bio-One, Kremsmünster, Austria, #655074) were coated overnight at 4 °C with 0.5 μg/mL recombinant human CD40 (R&D Systems, Minneapolis, MN, USA, #1493-CDB) in PBS.

Techniques: In Vivo, Transgenic Assay, MANN-WHITNEY

Journal: Science Advances

Article Title: STAT3 haploinsufficiency is associated with autosomal dominant hyper-IgE syndrome

doi: 10.1126/sciadv.adw2464

Figure Lengend Snippet: ( A ) STAT3 expression was evaluated by Western blot in Stat3 +/+ and Stat3 +/− cells derived from the spleen, thymus, and bone marrow (BM). Tubulin was used as loading control. Each lane represents one mouse. ( B ) A quantification of the STAT3/loading control ratio after normalization is shown for independently performed experiments as in (A). The mean ± SD is plotted. ( C ) IgE serum levels in Stat3 +/+ and Stat3 +/− mice were measured by ELISA. ( D ) Flow cytometry to identify IgG1 + and IgE + cells from IgM − IgD − cells 4 days after culturing Stat3 +/+ and Stat3 +/− B220 + splenocytes with IL-4 (12.5 ng/ml) and anti-CD40 antibody (62.5 ng/ml) in the absence or presence of IL-21 (25 ng/ml). Numbers in dot plots represent cell frequencies. ( E ) Mean (±SD) frequencies of the IgG1 + and IgE + cells as gated in (D). ( F ) Levels of IgG1 and IgE in the supernatant of cell cultures as done in (D) were measured by ELISA. B220 + splenocytes from Stat3 +/+ and Stat3 +/− mice were stimulated for different time points with IL-21 (50 ng/ml) ( G ), IL-10 (50 ng/ml) ( H ), and IL-6 (50 ng/ml) ( I ), and the phosphorylation of STAT3 (p-STAT3) was evaluated by flow cytometry. Mean (±SD) of mean fluorescent intensity (MFI) is plotted. Data are representative of at least three independent experiments. P values by unpaired t test (C) or two-way ANOVA followed by Bonferroni’s or Tukey’s multiple comparisons test (B and E to I).

Article Snippet: Cells were cultured in complete B cell medium [RPMI with 10% FBS, 10 mM Hepes, GlutaMAX, penicillin-streptomycin (Gibco), and 50 μM β-mercaptoethanol (Sigma-Aldrich), supplemented with IL-4 (12.5 ng/ml; PeproTech) and anti-CD40 antibody (62.5 ng/ml; Miltenyi Biotec), in the absence or presence of IL-21 (25 ng/ml; PeproTech)].

Techniques: Expressing, Western Blot, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Phospho-proteomics