CFU-O assay protocol (flushed) (by Masanobu Ohishi)
Materials
Medium; alpha MEM (GIBCO, Cat#32561-037) ; 500ml
20% FBS (HYCLONE; Cat# SH30070.03);100cc
Antibiotic-Antimycotic (Gibco:Cat#. 15240-062);6cc
Plate: FALCON 35-3846
25G needle
Scalpel (No.10) * Feather is the best!
ascorbic acid (Sigma #A4403-100MG)
*dissolve in alphaMEM(-FBS) (5mg/ml) and keep the aliquots in -20.
Glycero2-phosphate disodium salt hydrate (Sigma #G6251-100G)
*dissolve before use (0.454g/2cc ddH2O)
Procedure
1. Sacrifice mice and take the bilateral tibias and femurs and put them in HBSS on ice. Leave the muscle attached to the bones.
2. (In the hood) Dip the samples quickly in 70% EtOH and then put them in PBS. Remove the muscles.
3. (Under the microscope) Remove the rest of the connective tissues using scalpels. Then cut off the both metaphysis: (Tibia) Insertion of Patella tendon and Distal T-F joint. (Femur) Slightly distal to the capsule attachment and under the Minor Trochanter.
4. (In the hood) Put a cell strainer on a 50cc Falcon tube.
5. Flush the bone marrow with HBSS. (10cc/one bone)
6. Centrifuge the tubes at 1200rpm for 5 min.
7. Resuspend the cells into media. (WT:4 cc, PPR*Tg:2 cc)
8. Count the cell numbers
15ml acetic acid + (3ml trypan Blue ) + 15ml Cell suspension
Put it on ice for 5-10 min.
Put 10ml into 90ml PBS
Count the cell No.
You usually get
3.6-5.0 X 107 /mouse from a WT
1.6-3.00 X 107 /mouse from a PPR*Tg
9. Seed the cells on 6-well plate ( 8X106/cc X 2cc/well)
10. Incubate for 24 hrs
11. (day1) Wash the wells by rocking. Change to fresh HBSS 5 times. Incubate in osteogenic media for 3 days
12. (day4)Wash the wells by rocking. Change to fresh HBSS 3times. Incubate for 3 days
13. (day7) Wash the wells by rocking. Change to fresh HBSS 1 time. Incubate for 21 days
14. Von kossa Staining after 21 days
Updated 1/25/11 by RK