Protocol CFDAse

Protocol CFDAse References CFDAse

 

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CFDAse staining protocol

Chemicals:

bullet Carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDAse); dissolve in DMSO to 5 mmol/L, aliquot in 50 Ál and freeze in -20░C until use.
bullet Bovine Serum Albumin (BSA)
bullet PBS / 1% BSA 
bullet Phosphate Buffered Saline (PBS)

 

  1. Wash cells 3x in PBS to remove proteins.

  2. Aliquot cells at a concentration of 1.107 cells/ml in a 15 ml tube.

  3. Dilute CFDAse stock 1 : 10,000 to 0.5 Ámol/L in PBS from the 5 mmol/L stock. 

  4. Spin cells down, remove supernatant, and add 1 ml 0.5 Ámol/L CFDAse solution per 1.107 cells. Resuspend cells well. Incubate 5 minutes at RT.

  5. Wash the cells 2x with PBS / 1% BSA at 4░C to stop the labelling.

  6. Cells can be used for assay now.

NB.   Due to high intensity of the signal after labelling. This can be checked best the next day.
NB2. The population of undivided cells should be identified by culturing a portion of the same cells for the same period without activation.

 

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 Suggestions, literature and protocols are welcome at icstaining@schuitemaker.info. Copyright 2000 - 2005.
Page was last updated: 05-03-2005