Journal: bioRxiv

Article Title: Initiation of primary T cell—B cell interactions and extrafollicular antibody responses in an organized microphysiological model of the human lymph node

doi: 10.1101/2025.01.12.632545

Figure Lengend Snippet: (a-c) Naïve T cells were cultured on chip with IL-7. Representative image (b) and quantification (c) of T cell viability after 4 days, labeled with (Calcein AM, green, and Dapi, blue) for 3 donors. (d-g) A CCL21 gradient was established on chip, with CCL21 added to the left-hand media lane. Representative images (e) and quantification (f) of naive CD4+ T cells after migrating toward CCL21 for 1 hr and staining with Calcein AM (green). (g) Quantification of cell velocity 30 min after gradient set up (cells were unlabeled). (h-k) Naïve T cells were cultured without (i) and with (ii) a-CD3/CD28 (StemCell). Images of CD69+ signal (FITC-anti-CD69, green) for (i) naïve and (ii) activated T cells on-chip, and quantification (j) of CD69 signal after 48 hours (unpaired T test, **:p<0.005). (k) Quantification of IFN-γ secretion by activated or naïve CD4+T cells on-chip measured by ELISA of supernatants collected at day 5. Panels f, g, k analyzed with ordinary two-way ANOVA with Sidak’s multiple comparisons test w/single pooled variance, ns: p>0.05, *:p<0.05, ****:p<0.00005.

Article Snippet: IgM was detected using a Human IgM ELISA Antibody Pair Kit (StemCell technologies, Cat#01995), and IL-21 was detected using an ELISA Flex: Human IL-21 (HRP) kit (Mabtech, Ohio, USA, Cat#3540–1H-6) following manufacturer protocols.

Techniques: Cell Culture, Labeling, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Initiation of primary T cell—B cell interactions and extrafollicular antibody responses in an organized microphysiological model of the human lymph node

doi: 10.1101/2025.01.12.632545

Figure Lengend Snippet: (a) Schematic of experimental setup for comparing naïve T versus pre-Tfh cells and presence/absence of SEB on IgM secretion. (b) ELISA data showing IgM secretion, dots represent pooled supernatant from four chips per donor. (c) Schematic of experimental setup for analyzing IgM secretion dependency on proximity between pre-Tfh and activated B cells. (d) Brightfield images showing patterned lymphocytes on chip, day 1. (e) ELISA data showing IgM secretion. Results from four pooled chips from one donor, D68F. Red dotted lines are ELISA limit of detection: 0.140 ng/mL IgM.

Article Snippet: IgM was detected using a Human IgM ELISA Antibody Pair Kit (StemCell technologies, Cat#01995), and IL-21 was detected using an ELISA Flex: Human IL-21 (HRP) kit (Mabtech, Ohio, USA, Cat#3540–1H-6) following manufacturer protocols.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Initiation of primary T cell—B cell interactions and extrafollicular antibody responses in an organized microphysiological model of the human lymph node

doi: 10.1101/2025.01.12.632545

Figure Lengend Snippet: (a) Schematic of experimental setup. (b) Representative composite images (fluorescence overlayed over brightfield) from D69M, cells stained with AF546-anti-CD38 (cyan) on day 7. (c) Change in %CD38 positive area (3 donors). (d) IgM secretion from co-culture, quantified by ELISA on day 6. N=2–3 chips/donor (5 donors). Analyzed using two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: IgM was detected using a Human IgM ELISA Antibody Pair Kit (StemCell technologies, Cat#01995), and IL-21 was detected using an ELISA Flex: Human IL-21 (HRP) kit (Mabtech, Ohio, USA, Cat#3540–1H-6) following manufacturer protocols.

Techniques: Fluorescence, Staining, Co-Culture Assay, Enzyme-linked Immunosorbent Assay