Live-cell imaging of the plasma membrane of Jurkat T cells
Ezra Bruggeman, Markus Körbel
Abstract
This is a protocol for the preparation of a Jurkat T cells for live imaging of the plasma membrane. This protocol was used to generate the data shown in Figure 6 of the following publication:
- Bruggeman et al., POLCAM: Instant molecular orientation microscopy for the life sciences. bioRxiv 2023.02.07.527479 (Feb 2023), doi: http://doi.org/10.1101/2023.02.07.527479
Steps
Cell culture
J8 LFA-1 cells were incubated overnight (approximately 18h 0m 0s) in complete-RPMI (StableCell RPMI-1640 media, Sigma) supplemented with 10 % (v/v) fetal calf serum (FCS), 1 % (v/v) HEPES buffer, and 1 % (v/v) pen/strep antibiotics.
1mL of cells were collected by centrifugation and resuspended in phenol-red free RPMI supplemented with 1 % HEPES.
Cell Labeling
Round coverslips were rinsed with IPA, MilliQ, dried, and Ar-plasma cleaned for 0h 20m 0s.
Grace Bio-Labs CultureWells were attached, and the slide was incubated with OKT3 antibody (provided by the Human Immunology Unit, WIMM, Oxford) for 0h 30m 0s.
The slide was washed 5 times with phenol-red free RPMI supplemented with 1 % HEPES.
Perform a final wash with phenol-red free RPMI supplemented with 1 % HEPES and 200 nM NR4A (MemGlowTM NR4A Membrane Polarity Probe, Cat. #MG06, Cytoskeleton Inc.) before imaging.
