18 mm glass coated surfaces

Jurkat T cells (ATCC Cat# TIB-152, RRID:CVCL_0367) and flotillin-1 and flotillin-2 knock-out Jurkat cell lines were cultured in RPMI 1640 medium (Gibco) supplemented with 10% (vol/vol) FBS, 2 mM l-glutamine and PenStrep (all from Invitrogen). Flotillin-1 and flotillin-2 knock-out Jurkat cell lines were generated using two guide RNAs each targeting the genomic DNA of flotillins 1 and 2 together with Cas9 expression plasmid. Transfected single cells were FACS sorted and seeded into 96-well plates and screened for flotillin1/2 knockout by western blot^{14 (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) that were prepared by incubating with poly-l-lysine (Sigma) for 30 min at room temperature, then 1 µM anti-CD3ε (16-0037; eBioscience) and anti-CD28 (16-0289; eBioscience) antibodies for T cell activation. For live cell imaging, cells were imaged from 10 to 40 min after their deposition on the coverslips.
For immunostaining, cells were fixed with 3.7% EM-grade paraformaldehyde (C004, ProScitech) for 30 min at 37 °C. After fixation, cells were permeabilized with 0.15% triton-X100 (Sigma), blocked in 5% BSA and probed with primary and secondary antibodies sequentially.

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.