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. Author manuscript; available in PMC: 2023 Jun 23.
Published in final edited form as: J Immunol Methods. 2022 Jan 21;502:113228. doi: 10.1016/j.jim.2022.113228

Fig. 1. CellTrace detection of proliferated T cells and flow cytometry gating.

Fig. 1

A. Scheme of mixed leukocyte reaction (MLR) with CellTrace pre-loading of PBLs on day 0 prior to co-culturing with moDCs. MoDCs and PBLs from the same donor (autologous, Auto) pairs were used as negative controls for T cell activation. Pairs with PBLs and moDCs from different donors (allogenic, Allo), were used to initiate T cell proliferation/activation, which was detected by flow cytometry and ELISA. B. Flow cytometry gating strategy used for the MLRs or for the PBLs activated with anti-CD3 anti-CD28 beads (+/-Beads). The cells were selected based on forward and side scatter, followed by exclusion of cell clumps (single cells). Next, eFluor780 staining was used to exclude dead cells. Live cells were analyzed for CD3, CD40L and CellTrace. A reduction of intensity in the CellTrace histograms indicates proliferated cells. Arrows show gating order. Experiments were performed with cells isolated from 4 donors; quantification in Fig. 3.