University:
Boston University
Course:
SAR HP 531 | Clinical Medicine I
Academic year:

2023
Views:

278

Pages:

1
Author:

Brendon Freeman

CFSE Staining CFSE binds to proteins; stain cells in buffers that have decreased serum concentration (0.1% FBS). Cells which incorporate too much CSFE will have reduced protein function and therefore reduced viability. A short efflux step using 100% serum is included to allow cells to “purge” some CFSE and washing the cells at least 3 times helps produce a tighter initial CFSE peak. CFSE should be prepared in DMSO and stored in small aliquots at -80. It decays quickly, yellow discoloration indicates the CFSE will no longer conjugate to proteins. PROTOCOL (optimized for human lymphocytes): Label: Cell suspension should be 20 million cells per ml in 0.1% FBS/PBS. Add CFSE to a final concentration of 1.5uM, vortex gently and let suspension sit for 8 min at room temp. (Larger cells will require more CFSE as will phagocytic cell types. Some researchers use as much as 50uM.) Stop Label and Efflux: Add an equal volume of pre-warmed FBS (100%, filtered). Incubate cells in a 37o water bath for 10 minutes for efflux. Wash: Centrifuge cells 5min, 400g. Discard supernatant and vortex pellet to obtain single cell suspension. Wash cells by suspending in l ml of 2%FBS/PBS for every million cells. Centrifuge cells 5 min, 400g. Discard supernatant and vortex pellet to obtain single cell suspension. Repeat wash 3 times. Take a day zero CSFE sample to determine initial labeling. Run cells immediately or fix in 14% paraformaldehyde. Culture: Culture cells in appropriate growth media and take samples as determined by your protocol. Run cells immediately or fix in 1-4% paraformaldehyde. Stain: Stain for surface markers at this point. When planning to surface stain, take into consideration that CSFE reads on the Flow Cytometer as FITC. Run cells immediately or fix in 1-4% paraformaldehyde.

CFSE Staining Protocol 1

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