May 09, 2024

Public workspaceLive-cell imaging of the plasma membrane of Jurkat T cells

Nature Methods
DOI
dx.doi.org/10.17504/protocols.io.kxygxyzxzl8j/v1
  • 1University of Cambridge
Ezra Bruggeman
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DOI: dx.doi.org/10.17504/protocols.io.kxygxyzxzl8j/v1
External link: http://ttps://doi.org/10.1101/2023.02.07.527479
Protocol Citation: Ezra Bruggeman, Markus Körbel 2024. Live-cell imaging of the plasma membrane of Jurkat T cells. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxyzxzl8j/v1
Manuscript citation:
Bruggeman et al., POLCAM: Instant molecular orientation microscopy for the life sciences. bioRxiv 2023.02.07.527479 (Feb 2023), doi: https://doi.org/10.1101/2023.02.07.527479
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 09, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 99508
Keywords: ASAPCRN, Jurkat T cell, POLCAM, NR4A, Microscopy
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000509
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:

Cell culture
Cell culture
J8 LFA-1 cells were incubated overnight (approximately Duration18:00:00 ) 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.

18h
Amount1 mL of cells were collected by centrifugation and resuspended in phenol-red free RPMI supplemented with 1 % HEPES.

Cell Labeling
Cell Labeling
50m
50m
Round coverslips were rinsed with IPA, MilliQ, dried, and Ar-plasma cleaned for Duration00:20:00 .

20m
Grace Bio-Labs CultureWells were attached, and the slide was incubated with OKT3 antibody (provided by the Human Immunology Unit, WIMM, Oxford) for Duration00:30:00 .

30m
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 (MemGlow‱ NR4A Membrane Polarity Probe, Cat. #MG06, Cytoskeleton Inc.) before imaging.