Jurkat T cells (Clone E6.1) and flotillin-1 and flotillin-2 knock-out Jurkat cell lines were cultured in
RPMI 1640 medium (Pan Biotech) supplemented with 10% (vol/vol)
FCS (Gibco). Flotillin-1 and flotillin-2 knock-out Jurkat cell lines were generated as described in [15 (
link)]. Cells were transfected with 1 μg DNA per 200,000 cells, 18–20 h prior to imaging using the
Neon electroporation kit (Invitrogen).
Before imaging, cells were incubated for 10 min at 37 °C on
18-mm glass-coated surfaces (Marienfeld, #0,117,580) that were prepared by incubating with
poly-L-lysine (Sigma, #P8920) for 30 min at room temperature, then 10 μg/ml
anti-CD3ε (eBioscience, #16–0037) and
anti-CD28 (eBioscience, #16–0289) antibodies for T cell activation. For live cell imaging, cells were imaged from 10 to 40 min after their deposition on the coverslips.
For imaging transferrin internalisation, transfected Jurkat T cells were activated for 5 min, transferred onto ice, media exchanged for fresh cold media with 25 μg/ml transferrin-Alexa488 (Jackson ImmunoResearch, #009–540-050) or
transferrin-Alexa647 (Jackson ImmunoResearch, #009–600-050) added, incubated for 5 min, media exchanged twice with fresh cold media, then transferred to 37 °C and imaged.
Rossatti P., Redpath G.M., Ziegler L., Samson G.P., Clamagirand C.D., Legler D.F, & Rossy J. (2022). Rapid increase in transferrin receptor recycling promotes adhesion during T cell activation. BMC Biology, 20, 189.