Figure 2
From: Expamers: a new technology to control T cell activation
Expamers are a potent T cell activation reagent. (A) Graph shows changes in WST-1 reagent depicted as arbitrary units 48 h after activation with either Expamers or Dynabeads from four independent experiments. Bars represent mean ± SD. Difference between the two stimulation conditions was not significant (two-tailed paired Student’s t test). (B) Human primary T cells were incubated at 37 °C with OKT3 monoclonal anti-CD3 antibody (positive control), anti-CD3 Fab-loaded Expamers in the presence or absence of D-biotin or anti-CD3 Fab fragments only (negative control) for the indicated timepoints. Cells were lysed and cytoplasmic extracts were analyzed for ZAP70 kinase phosphorylation using SDS-PAGE and Western Blot. GAPDH was used as loading control and anti-Strep-Tag as well as anti-heavy chain secondary antibodies were used for reagent detection. One representative blot of three independent experiments is shown. Full-length blots/gels are presented in Supplementary Fig. S8. (C) Histograms from one representative experiment from four independent measurements shows changes in fluorescence intensity of tdTomato-Nur77 reporter in a Jurkat T cell-line over time upon activation with Expamers. For Dynabeads control, 24 h time point is shown. (D) Histograms from one of two independent experiments represent changes in DNA content over time upon Expamers activation. (E) CD69 and CD25 surface marker upregulation was detected 24 h after stimulation with Expamers using flow cytometry. T cells were pre-gated on live, single CD3-positive cells. One representative dot plot is shown. Graph summarize data from 8 independent measurements. Bars represent mean ± SD. (F) Histograms present cell cycle entry and proliferation that were assessed by measuring CFSE dilution of CFSE-labeled T cells by flow cytometry at day 3 and day 7 time point. Numbers on top of the histograms refer to the number of cell division within each generation. (G) T cell proliferation is represented by fold expansion graph that is a cumulative data from at least four experiments. Negative control is represented by unstimulated cells. Lines represent mean ± SD. Difference between two stimulation conditions was not significant (two-tailed paired Student’s t test).
