Transferrin alexa647

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.

Jurkat T cells were collected and re-suspended in serum-free RPMI containing 30 μg/ml transferrin-Alexa647 (Jackson ImmunoResearch, #009–600-050) and supplemented (activated) or not (resting) with 1.5 μg/ml anti-CD3ε (OKT-3) + 1 μg/ml anti-CD28 (CD28.2). Cells were then incubated for 0 min or 30 min at 37 °C, cooled down on ice and washed twice with cold FACS buffer (PBS + 2% FCS) to remove unbound transferrin-Alexa647. Subsequently, fluorescence of at least 1 × 10⁴ cells per sample was analysed at a LSR II flow cytometer (BD Biosciences). Samples were kept on ice at all times before measuring. SYTOX Blue (0.1 μM; Invitrogen, #S11348) was added directly before measuring for identification of dead cells. The ratio of MedianFI of activated/resting cells was calculated for 0 min and 30 min timepoints.
To assess efficacy of anti-TfR treatment, Jurkat and primary T cells were treated or not with 10 μg/ml anti-TfR (M-A712) and activated for 20 h on plate-bound anti-CD3ε (OKT-3) and anti-CD28 (CD28.2). transferrin-Alexa647 (30 μg/ml) was added for the last 90 min of incubation. Subsequently, activated cells were re-suspended, washed twice with PBS and at least 1 × 10⁴ cells per sample were analysed using a LSR II flow cytometer (BD Biosciences).