A1r tie live cell imaging confocal system

Cells were grown to ≈ 80 % confluency on glass-bottomed, 12-well plates (MatTek, P12G-1.5–14-F). After the culture medium had been aspirated, the cells were washed three times with prewarmed Dulbecco’s phosphate-buffered saline (DPBS; Life Tech, 14040117) and incubated with the indicated concentration of NR₇₄₄, along with the indicated subcellular organelle tracker, for 30 min in culture medium. Cells were then washed with DPBS three times and imaged in DPBS. Confocal microscopy was performed with a Nikon A1R-TiE live-cell imaging confocal system. Laser lines used include blue (405 nm), green (488 nm), and far-red (640 nm). ImageJ software was used for data analysis.

To visualize T_FH and T_FR cells in lymph node tissues, immunofluorescence staining was conducted according to a previously described method (38 (link)). Goat polyclonal anti-human PD-1 Abs (catalog number AF1086, 1:100; R&D Systems), rabbit monoclonal anti-human CD4 Abs (clone EPR6855, 1:200; Abcam), and mouse monoclonal anti-human Foxp3 Abs (clone 236A/E, 1:200; Abcam) were incubated with tissues sections. Following primary Ab incubation and washing, donkey anti-goat IgG conjugated with Alexa Fluor 488 (catalog number A-11055, 1:100), donkey anti-rabbit IgG conjugated with Alexa Fluor 594 (catalog number R37119, 1:100), and donkey anti-mouse IgG conjugated with Alexa Fluor 647 (catalog number A-31571, 1:100; all from Thermo Fisher Scientific) were used. Cell nuclei were counterstained with DAPI. A Nikon A1R-TiE live cell imaging confocal system was used to visualize and capture images of stained samples.