FACS Analysis (Cells from CFU Culture)

FACS analysis (crushed BM only) by Masanobu Ohishi

Materials

1X DPBS (Cellgro, #21-031-CV) FBS (Hyclone, #SH30070.03) HBSS (Gibco, #24020) mortar and pestle Cell strainers (BD Falcon #352350) type 1 collagenase (Worthington biochemical LS004194) type 2 collagenase (Worthington biochemical LS004174) FACS tube (BD Falcon #352054) FACS tube with filter (BD Falcon #352235) Ficoll-Paque (GE Healthcare, #71-7167-00 AG)

Antibodies See antibody binder to plan experiment accordingly.

Preparation

 Dilute the antibodies in DPBS (2%FBS) (1/250) o For instance, for a single stain: 1 ml antibody / 250 ml DPBS (2%FBS) o For a triple stain, 1 ml each antibody in 250 ml DPBS (2%FBS)  Dissolve collagenase (weigh in advance, dissolve just before digestion): o (for periosteum) Collagenase1 0.00625g and Collagenase2 0.01875g in 10ml CaCl2(2.5mM) + 15ml alphaMEM(-FBS) (Gibco, Cat#32561); or dissolve in 25 ml HBSS. Prewarm CaCl2/media or HBSS to 37 degrees prior to dissolving. Use 8-10ml/mouse.  FACS tubes with antibodies o For single staining 50 ml /tube o For the samples (> 50 ml)

Procedure

1. Isolate the hind limbs and iliac crests. and clean the bones with gauze strips 2. Scrape periosteum with scalpel blade 3. Collagenase digestion (15min, 37degree) and scrape again with scalpel 4. Detach the epiphysis with scissors

BM 5. Cut the bones in 3-4 pieces and crush the bones in 4ml DPBS (2%FBS) 6. Collect the supernatant and pour into falcon tubes through cell strainer. 7. Add 2 ml DPBS (2%FBS) to mortar, rinse fragments, and collect liquid and add to supernatant through cell strainer.

Updated 1/25/11 by RK 8. Add the cell suspension into 3 ml Ficoll gradient (tilt the tubes and pipet the cell suspension slowly on the wall of the tubes) 9. Spin down (10th floor Beckman GP; 1800 rpm, 10 min. breaker off) 10. Draw the lines above and below the cell fraction line. Aspirate the sup to the upper line then collect the cell fraction (around 2.5cc) and pour into 50cc Falcon tube 11. Add DPBS(2% FBS) to have total volume of 10 cc. 12. Count the cells 13. Spin down (11th floor tissue culture Sorvall GLC-2B, 1200rpm for 10min) and adjust the cell concentration to 5X10*7 /ml

Antibody incubation 14. Incubate the cells with antibody mix. For the single stains, 1:1. For example, 50 ml cells: 50 ml Ab dilution. For the samples, 100ml: 100 ml or more 15. Add X9 volume of DPBS(2% FBS) (e.g. if the cell antibody mix is 200 ml in total, add 1800 ml of PBS ) 16. Spin down (11th floor, Beckman J-6M, 8 degree, 900 rpm, 5 min.) 17. Repeat wash and spin. 18. Aspirate the supernatant and resuspend the cell pellets in 300 ml DPBS (2% FBS). 19. Analyze.

Updated 1/25/11 by RK