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BrdU FACS

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					BrdU/ PI FACS protocol
BrdU Flow Kit BD Pharmingen cat no 559619


   1. Labeling of cells with BrdU. Label cells with 10 µM BrdU for 45 min. Stock
       solution of BrdU stored at –80 ˚C is 10 mg/ml. Dilute to 1 mM by taking 31 µl
       stock and adding PBS up to 1 ml total volume (32x dilution). Add 100 µl of the
       diluted 1 mM stock to each p100 dish with 10 ml medium. Remember a negative
       control (no BrdU added!)
   2. Collect cells by treating them with PBS 0.1 % EDTA for 5 min at 37 ˚C. Use a
       Pasteur pipet to generate a homogenous population. Transfer to 15 ml Falcon
       tube.
   3. Fixing and permeabilizing: Resuspend cells in 100 µl of BD Cytofix/ Cytoperm
       Buffer per tube. Incubate cells for 15-30 min on ice. Wash cells 1x with 1 ml of
       1x BD Perm/ Wash buffer.
   4. Resuspend cells in 100 µl of BD Cytoperm Plus Buffer per tube. Incubate 10
       min on ice. Wash cells 1x with 1ml 1x BD Perm/ Wash buffer.
   5. Refixation: Resuspend cells in 100 µl of BD Cytofix/ Cytoperm Buffer per tube.
       Incubate 5 min on ice. Wash cells 1x with 1ml 1x BD Perm/ Wash buffer.
   6. DNase treatment: Resuspend in 100 µl of diluted DNase (300 µl of 1 mg/ml
       DNase + 700 µl PBS to make a stock of 300 µg/ml)
   7. Incubate 1 hr at 37 ˚C
   8. Wash cells 1x with 1 ml of 1x BD Perm/ Wash buffer.
   9. Stain BrdU: Resuspend cells in 50 µl of BD Perm/ Wash Buffer containing
       FITC anti-BrdU (1 µl stock into 50 µl)
   10. Incubate 20 min at room temperature (wrapped in metal foil to protect from light)
   11. Wash cells 1x with 1 ml of 1x BD Perm/ Wash buffer.
   12. PI staining: Make up the following PI staining solution: 50 µg/ml PI, 10 mM Tris
       pH 7.5, 5 mM MgCl2, 10 µg/ml RNase (guaranteed DNase free). For 10 ml, mix
       the following: 1 ml 0.5 mg/ml PI stock soln., 100 µl 1 M Tris pH 7.5, 50 µl 1 M
       MgCl2, 20 µl RNase A, 8.9 ml dH2O
   13. Add 0.5 ml of PI staining solution for each sample (if using cells from a p100
       dish; when using a p60 dish, 0.5 ml is sufficient) and mix by pipetting up and
       down. Incubate at 37 ˚C for 30 min. The cells are now ready for analysis and
       should be analyzed within 24 hrs. Until analysis, keep the cells on ice with the
       tubes wrapped in aluminium foil, since PI is light sensitive. You have to transfer
       your cells in the end to Falcon 2054 tubes !
   PI stock soln. is 10x: 0.5 mg/ml PI in 0.038M citrate pH 7.0 (stored at 4 ˚C)

				
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posted:8/8/2011
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