Journal: Science Advances

Article Title: P2X4 and P2X7 are essential players in basal T cell activity and Ca 2+ signaling milliseconds after T cell activation

doi: 10.1126/sciadv.abl9770

Figure Lengend Snippet: Ca 2+ imaging of CD4 + T cells of WT or P2rx4 −/− and P2rx7 −/− mice. Cells were stimulated with anti-CD3/anti-CD28–coated beads. A minimum of 10 different mice were used. (B to D) Data are means ± SEM; WT, n = 46 cells; P2rx4 −/− , n = 47 cells; P2rx7 −/− , n = 39 cells. Statistical analysis by Kruskal-Wallis test. ( A ) Representative cells of WT or P2rx4 −/− and P2rx7 −/− were shown for 0.52 s before and up to 15 s after stimulation with anti-CD3/anti-CD28–coated beads (scale bar, 5 μm) as well as for 0 to 0.65 s in 0.13-s steps zoomed into the region of bead contact (scale bar, 1 μm). ( B ) Quantification of the first 15 s after bead contact for CD4 + T cells of WT or P2rx4 −/− and P2rx7 −/− mice. The percentage of responding cells, the number of Ca 2+ microdomains per frame for whole cells (confocal plane), and the average Ca 2+ concentration of these signals are shown. ( C ) Quantification of the number of Ca 2+ microdomains per frame for the period 2.5 to 0 s before and every 5 s after bead contact up to 25 s after bead contact. ( D ) Analysis of the Ca 2+ microdomains in the first second before and after TCR stimulation for the sublayers at the contact site (as indicated in red) (left). Quantification of the signals in the first second after bead contact (right). * P < 0.05, ** P < 0.005, *** P < 0.001.

Article Snippet: The monoclonal antibodies (mAbs) anti-mouse CD3 and anti-mouse CD28 were obtained from BD Biosciences.

Techniques: Imaging, Concentration Assay

Journal: Science Advances

Article Title: P2X4 and P2X7 are essential players in basal T cell activity and Ca 2+ signaling milliseconds after T cell activation

doi: 10.1126/sciadv.abl9770

Figure Lengend Snippet: Ca 2+ imaging of CD4 + T cells of WT mice incubated with or without PSB-15417 (1 μM) for the inhibition of P2X4 or inhibiting nanobody (P2X7-Nb) 13A7-dim-Alb (1 μg/ml) for the inhibition of P2X7. A dimethyl sulfoxide (DMSO) control (0.01%) or an irrelevant nanobody (Control Nb; dummy-dim-Alb, 1 μg/ml) was used. Cells were stimulated with anti-CD3/anti-CD28–coated beads. A minimum of five different mice were used. (B to D) Data are means ± SEM; DMSO control, n = 28 cells; control nanobody, n = 40; cells treated with PSB-15417, n = 29 cells; cells treated with the P2X7-inhibiting nanobody, n = 30 cells. Statistical analysis by an unpaired two-tailed Mann-Whitney test. ( A ) , inhibited P2X4 and P2X7 channels were shown for 0.52 s before and up to 15 s after stimulation with anti-CD3/anti-CD28–coated beads (scale bar, 5 μm) as well as for 0 to 0.65 s in 0.13-s steps zoomed into the region of bead contact (scale bar, 1 μm). ( B ) Quantification of the first 15 s after bead contact for CD4 + T cells of controls or inhibited P2X4 and P2X7. ( C ) Quantification of the number of Ca 2+ microdomains per frame for the period 2.5 to 0 s before and every 5 s after bead contact up to 25 s after bead contact. DMSO control and cells treated with PSB-15417 were compared (left), as well as the control Nb with the P2X7-Nb (right). ( D ) Analysis of the Ca 2+ microdomains in the first second before and after TCR stimulation for the sublayers at the contact site (as indicated in red) (left). Quantification of the signals in the first second after bead contact (right). * P < 0.05, ** P < 0.005, *** P < 0.001.

Article Snippet: The monoclonal antibodies (mAbs) anti-mouse CD3 and anti-mouse CD28 were obtained from BD Biosciences.

Techniques: Imaging, Incubation, Inhibition, Two Tailed Test, MANN-WHITNEY

Journal: Science Advances

Article Title: P2X4 and P2X7 are essential players in basal T cell activity and Ca 2+ signaling milliseconds after T cell activation

doi: 10.1126/sciadv.abl9770

Figure Lengend Snippet: Ca 2+ imaging of CD4 + T cells of WT mice incubated with or without 10 U of apyrase. Cells were stimulated with anti-CD3/anti-CD28–coated beads. A minimum of five different mice were used. (B to D) Data are means ± SEM; WT, n = 31 cells; cells treated with apyrase, n = 27 cells. Statistical analysis was done using a nonparametric unpaired two-tailed Mann-Whitney test. ( A ) Representative cells of WT or cells treated with apyrase were shown for 0.52 s before and up to 15 s after stimulation with anti-CD3/anti-CD28–coated beads (scale bar, 5 μm) as well as for 0 to 0.65 s in 0.13-s steps zoomed into the region of bead contact (scale bar, 1 μm). ( B ) Quantification of the first 15 s after bead contact for CD4 + T cells of WT or cells treated with apyrase. The percentage of responding cells, the number of Ca 2+ microdomains per frame for whole cells (confocal plane), and the average Ca 2+ concentration of these signals are shown. ( C ) Quantification of the number of Ca 2+ microdomains per frame for the period 2.5 to 0 s before and every 5 s after bead contact up to 25 s after bead contact. ( D ) Analysis of the Ca 2+ microdomains in the first second before and after TCR stimulation for the sublayers at the contact site (as indicated in red) (left). Quantification of the signals in the first second after bead contact (right). * P < 0.05, ** P < 0.005, *** P < 0.001.

Article Snippet: The monoclonal antibodies (mAbs) anti-mouse CD3 and anti-mouse CD28 were obtained from BD Biosciences.

Techniques: Imaging, Incubation, Two Tailed Test, MANN-WHITNEY, Concentration Assay

Journal: Science Advances

Article Title: P2X4 and P2X7 are essential players in basal T cell activity and Ca 2+ signaling milliseconds after T cell activation

doi: 10.1126/sciadv.abl9770

Figure Lengend Snippet: Ca 2+ imaging of CD4 + T cells of WT mice incubated with or without 200 μM PANX1 inhibitor 10 panx1 20 min before measurements. Cells were stimulated with anti-CD3/anti-CD28–coated beads. A minimum of 10 different mice were used. (B to D) Data are means ± SEM; WT, n = 39 cells; cells treated with 10 panx1, n = 48 cells. Statistical analysis by an unpaired two-tailed Mann-Whitney test. ( A ) Representative cells of WT or cells treated with 10 panx1 were shown for 0.52 s before and up to 15 s after stimulation with anti-CD3/anti-CD28–coated beads for whole cells (scale bar, 5 μm) as well as for 0 to 0.65 s in 0.13-s steps zoomed into the region of bead contact (scale bar, 1 μm). ( B ) Quantification of the first 15 s after bead contact for CD4 + T cells of WT or cells treated with 10 panx1. The percentage of responding cells, the number of Ca 2+ microdomains per frame for whole cells (confocal plane), and the average Ca 2+ concentration of these signals are shown. ( C ) Quantification of the number of Ca 2+ microdomains per frame for the period 2.5 to 0 s before and every 5 s after bead contact up to 25 s after bead contact. ( D ) Analysis of the Ca 2+ microdomains in the first second before and after TCR stimulation for the sublayers at the contact site (as indicated in red) (left). Quantification of the signals in the first second after bead contact (right). ** P < 0.005, *** P < 0.001.

Article Snippet: The monoclonal antibodies (mAbs) anti-mouse CD3 and anti-mouse CD28 were obtained from BD Biosciences.

Techniques: Imaging, Incubation, Two Tailed Test, MANN-WHITNEY, Concentration Assay

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 1 Coadministration of CD3/CD28 and CpG induce robust amounts of IL-10 expression in splenocytes when compared to either signal alone. (A) Splenocytes were treated with CpG, CD3/CD28, CD3/CD28/CpG, or left untreated for 72 hours and analyzed for IL0 production by ELISA. (B) Splenocytes were treated with CD3/CD28/CpG for 0, 48, or 72 hours, and IL-10 levels were measured by ELISA. (C) Supernatants of splenocytes treated with either CpG or control ODN (ctrl CpG) in the absence or presence of CD3/CD28 for 72 hours were analyzed for IL-10 production by ELISA. (D) Splenocytes were treated with CpG in the presence or absence of various T cell-activating antibodies (CD28, CD3, or CD3/CD28) for 72 hours, and IL-10 expression in the supernatants was measured via ELISA. (E) Supernatants from splenocytes treated as indicated for 72 hours were analyzed for IL-10 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 2 Coordination between T cells and macrophages induces the highest expression of IL-10 in a cell-contact dependent manner. (A, B) Supernatants from wild type and nude (A, Balb/c background) or wild type and SCID (B, C3H background) splenocytes were treated with CD3/CD28/CpG for 72 hours and then analyzed for IL-10 production by ELISA. (C) Non-depleted splenocytes or the ones depleted for CD3, CD4, CD8, DC, and NK cells were treated with CD3/CD28/CpG for 72 hours, and the supernatants were analyzed for IL-10 expression via ELISA. (D) Purified splenic B cells, DC, CD4+ T cells, or peritoneal macrophages were coincubated in the presence or absence of purified CD4+ T cells, treated with CD3/CD28/CpG for 72 hours and analyzed for IL-10 expression in the supernatants by ELISA. (E) Peritoneal macrophages were coincubated in the presence or absence of purified NK, whole T (CD3+ T), CD4+ T, or a combination of CD4+ T and NK cells; treated with CD3/CD28/CpG for 72 hours; and analyzed for IL-10 expression in the supernatants via ELISA. (F) Peritoneal macrophages were coincubated with purified CD4+ cells in the presence or absence of a transwell barrier, treated with CD3/CD28/CpG for 72 hours, and analyzed for IL-10 expression in the supernatant via ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Purification

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 3 CD3/28/CpG induces IL-10 production in both macrophages and CD4+ T cells. (A) Diagram of FACS analysis. (B, C) Splenocytes left untreated or treated with CpG, CD3/CD28, CD3/CD28/CpG for 72 hours and number of CD4 (B) or F4/80 (C) were measured via FACS. (C, D) Median fluorescence intensity of IL10 in the CD4+ (D) or F4/80+ (E) gated cells. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Fluorescence

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 4 CD3/CD28/CpG regulates IL-10 via activation of NF-κB1,pERK, and STAT3. (A) Splenocytes were treated as indicated for 72 hours and IL-10 levels were measured by ELISA. (B) Supernatants from wild type or NF-κB1−/−splenocytes treated with CD3/CD28, CpG, or TX (CD3/CD28/CpG) for 72 hours were analyzed for IL-10 production by ELISA. (C) Peritoneal macrophages from wild type or NF-κB1−/−mice were coincubated with purified CD4+ T cells from either wild type or NF-κB1−/−mice, treated with CD3/CD28/CpG for 72 hours and the supernatants were measured for IL-10 expression by ELISA. (D) Splenocytes treated with CD3/CD28, CpG, or TX (CD3/CD28/CpG) for 72 hours were stained for CD4 and pERK or CD4 and pSTAT3. Histograms show pERK or pSTAT3 levels from gated T cells. (E) Quantification of median fluorescence intensity of pERK levels in CD4 cells (N = 3). (F) Splenocytes were treated with ERK inhibitor U0126 at various doses for 72 hours in presence of CD3/CD28/CpG, and IL-10 levels were measure by ELISA. (G) Splenocytes were treated with STAT3 inhibitor NSC 74859 at various doses for 72 hours in presence of CD3/ CD28/CpG, and IL-10 levels were measure by ELISA. (H) Wild type or NF-κB1−/−splenocytes in presence or absence of STAT3 inhibitor (STAT3i, 50 μM) were treated with CD3/CD28/CpG for 72 hours and analyzed for IL-10 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Purification, Expressing, Staining, Fluorescence

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 5 Activation or inhibition of CD40/CD154 pathway in the presence of CD3/CD28/CpG acts as a switch in the expression of IL-10 or IL-30. (A) Supernatants from wildtype or CD40−/−splenocytes were treated with CD3/CD28/CpG for 72 hours were analyzed for IL-10 production by ELISA. (B) Supernatants from wild type or CD154−/−splenocytes treated with CD3/CD28/CpG for 72 hours were analyzed for IL-10 production by ELISA. (C, D) Splenocytes from wild type mice were treated with the indicated treatments in the presence of control or anti-CD40 antibodies for 72 hours, and IL-10 (C) or IL-30 (D) expression was measured in the supernatants using ELISA. (E, F) Splenocytes from wild type mice were treated with the indicated treatments in the presence of control or anti-CD40 antibodies for 24, 48, or 72 hours, and IL-10 (E) or IL-30 (F) expression was measured in the supernatants using ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Activation Assay, Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 6 Regulation of IL-10 and IL-30 via activation of CD40 signaling. (A) Supernatants from splenocytes treated with CpG, CD3/CD28, TX (CD3/CD28/CpG), in the presence or absence of rIL-10 for 72 hours were analyzed for IL-30 production by ELISA. (B) Wild type or IL-10−/−splenocytes were treated as indicated for 72 hours, and IL-30 levels were measured by ELISA. (C) Splenocytes were treated with CD3/CD28/CpG in the presence of recombinant IL30 at 50 and 100 ng/ml for 72 hours, and IL-10 levels were measured by ELISA. (D) Wild type or IL-10−/−splenocytes were treated with CD3/CD28/CpG for 72 hours in the presence of control or anti-CD40 antibodies, and IL-30 levels were measured by ELISA. (E) Harvested pellets of splenocytes treated with anti-CD40 or control antibody for the indicated time points were probed for pSTAT3, p-p65, p50/105, and actin. (F) Splenocytes were treated with CD3/CD28/CpG in the presence or absence of anti-CD40 activating antibody and the levels of p-p65 and p-STAT3 in the CD4+ T cells or macrophages were measured via flow cytometry. (G) Supernatants from wild type or NF-κB1−/−splenocytes treated with CpG, CD3/CD28, TX (CD3/CD28/CpG), or CD3/CD28/CpG in the presence of activating anti-CD40 for 72 hours were analyzed for IL-30 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Control, Flow Cytometry

Journal: Cellular and Molecular Immunology

Article Title: Notch2−expressing CD4 + T cells attain immunoregulatory functions during autoimmune inflammation

doi: 10.1038/s41423-025-01318-2

Figure Lengend Snippet: Foxp3−expressing CD4 + Notch2 + T cells have immunomodulatory properties and exhibit a dysfunctional phenotype. Spinal cords were harvested 10−12 (P) or 21−25 (R) days after the induction of EAE. A RNA sequencing (RNA−seq) analysis of spinal cord−infiltrating CD4 + T cells was performed to evaluate the expression of representative RNAs encoding inflammatory factors and Treg cell−associated immunomodulatory factors. B Analysis of the correlation matrix via RNA sequencing (RNA−seq). C Representative flow cytometry analysis of IFN−γ + cell populations within different populations of spinal cord-infiltrating CD4 + T cells. D Quantification of the IFN−γ + cell populations via flow cytometry analysis (based on C ). The graph shows the means ± SDs of the percentages. E Representative flow cytometry analysis of the IL−17a + cell populations within different populations of spinal cord-infiltrating CD4 + T cells. F Quantification of the results of flow cytometry analysis of the IL−17a + cell populations (based on E ). G Quantification of the cytokine bead array (CBA) results showing the production of several cytokines after TCR stimulation in different populations of spinal cord−infiltrating CD4 + T cells. The graph shows the means ± SDs of cytokine secretion. H Representative flow cytometry analysis of congenic CD4 + T−cell proliferation upon coculture with spinal cord−infiltrating CD4 + T cells. I Quantification of congenic CD4 + T−cell proliferation upon coculture with spinal cord−infiltrating CD4 + T cells (based on H ). The graph shows the means ± SDs of high percentages of proliferating cells. J Representative flow cytometry analysis of proliferating CTV−labeled CD4 + T cells after stimulation for 72 h with anti−CD3 and anti−CD28 antibodies. K Representative flow cytometry analysis of protein expression (PD−1, Tim3, and Lag3) in CD4 + Notch2 + T cells from the spinal cords of EAE model mice. L Representative flow cytometry plots of MOG 38 − 49 −specific tetramer−binding CD4 + T cells from the spinal cords of EAE mice. The data are representative of two ( J ) or three ( C − Ι , Κ , L ) independent experiments (error bars, s.d. of > four ( D , F , G , I ) mice or samples). Statistical significance was calculated via one−way ANOVA followed by Tukey’s t−test for all the experiments; * P < 0.05, ** P < 0.01, *** P < 0.001. ns, not significant

Article Snippet: Anti−mouse CD3 (17A2) and anti−mouse CD28 (145−2C11) antibodies were purchased from Bio X Cell (Seoul, Korea).

Techniques: Expressing, RNA Sequencing, Flow Cytometry, Labeling, Binding Assay

Journal: Biomaterials

Article Title: Biphasic response of T cell activation to substrate stiffness.

doi: 10.1016/j.biomaterials.2021.120797

Figure Lengend Snippet: Fig. 1. Characterization of the hydrogel system for T cell activation. (A) Schematic of antibody coated hydrogels as T cell activation platform. Poly acrylamide gels were polymerized with streptavidin- acrylamide, then coated with biotinylated Goat-anti- mouse and secondary activating antibodies to CD3 and CD28. Mixed CD4+/CD8+ polyclonal primary human T cells were stimulated on activating sub strates. (B) Mechanical testing via indentation indi cate hydrogels with varying ratios of acrylamide and bis-acrylamide have Young’s modulus between 5 and 110 kPa (Data are mean ± s.d., n = 4 gels for 5 kPa, n = 7 gels for all other formulations). (C) Confocal microscopy imaging of fluorescent antibodies coated on hydrogels indicate attachment of antibodies to hydrogel surface. (D) Streptavidin-acrylamide con centrations were varied to obtain similar coating of antibodies on hydrogels, as verified by fluorescence intensities on the surface of gels. (n = 3 gels for each formulation).

Article Snippet: Key resources table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Biotinylated Goat-anti-mouse BioLegend 405303 Mouse-anti-human CD3 (OKT3) BioXCell BE0001-2 Mouse-anti-human CD28 (9.3) BioXCell BE0248 Biological Samples Leukopack New York Blood Center Chemicals, Peptides, and Recombinant Proteins Acrylamide (40%) Sigma 01697 Bis-acrylamide (2%) Fisher BP1404-250 Ammonium persulfate (APS) Sigma A3678 Tetramethylethylenediamine (TEMED) Sigma T7024 Streptavidin-acrylamide Fisher S21379 (3-Aminopropyl)triethoxysilane (APTES) Sigma 440140 Glutaraldehyde Sigma G7776 Alexa-fluor 488 Thermo A20000 Blebbistatin Sigma 203391 Prolong Diamond ThermoFisher P36961 Qiazol Qiagen 79306 Critical Commercial Assays Human IL-2 Secretion kit (APC) Miltenyi Biotec 130-090-763 RosetteSep Human T cell Enrichment Kit Stemcell Technologies 15061 CellTrace CFSE Dilution Assay ThermoFisher C34554 CellVue Jade ThermoFisher 88-0876-16 Software and Algorithms FlowJo 7.6 FlowJo LLC FCS Express V6 De Novo Software Graphpad Prism 7 Graphpad Software Kallisto Bray et al.

Techniques: Activation Assay, Confocal Microscopy, Imaging, Fluorescence, Formulation