Dear Zahra!

Please You look at following article and protocol:

Article RANKL-mediated osteoclast formation from murine RAW 264.7 cells

3.1. RAW 264.7 Cell Culture RAW 264.7 cells are obtained from the ATCC or similar cell repository. They represent a murine macrophage cell line that has the capability to be grown indefinitely as an OC precursor population or can be differentiated by treatment with RANKL into multinucleated bone pit resorptive OCs expressing the hallmark characteristics of in vivo formed OCs.)

3.2.1. Serum Gradient Purification of RAW-OC Because not all RAW cells fuse into multinucleated RAW-OCs by d 5 or 6, those that have can be purified from the remaining mononuclear cells using serum density gradient fractionation (see Note 8). This procedure is a modification of the one we routinely use to purify in vitro formed OC or OC-like cells from chick or human origin (17,18). Directions are provided for RAW-OCs formed on 100-mm tissue culture dishes. All steps are conducted at room temperature unless otherwise noted, and are performed in a sterile hood using sterile solutions and supplies.

1. Remove the spent culture medium from two 100-mm dishes of d 5 or 6 RANKLgenerated RAW-OCs.

2. Gently add 10 mL of MHB to each 100-mm dish to wash the cells. Remove and discard the washes.

3. Repeat step 2 to wash the RAW-OCs twice more with MHB.

4. Add 10 mL of MHB to each dish and place them into a tissue culture incubator at 37°C for 15 min.

5. Remove and discard the MHB solution from each dish.

6. Add 5 mL of freshly prepared collagenase–trypsin digestion solution to each dish and incubate at 37°C for 5 min.

7. Remove the dishes from the incubator and shake the plates gently by hand back and forth (e.g., slide the dish on a flat surface) for ~30 sec to detach and loosen the interaction of cells with extracellular matrix produced by the cells.

8. Completely remove the collagenase–trypsin solution containing the released matrix material from each dish and discard (see Note 9).

9. Gently wash the adherent cells on each dish by releasing 10 mL of PBS slowly against the side wall of the dish. Completely remove and discard the washes.

10. Repeat step 9 to wash the cells on each dish with PBS twice more.

11. Add 5 mL of protease–EDTA digestion solution to each dish. Incubate at 37°C for 10–15 min (see Note 10).

12. Loosen the adherent cells on each dish by flushing the protease–EDTA incubation solution with a pipet gently over the surface of the cell layer to free the cells (see Note 11).

13. Transfer the cell suspensions from two 100-mm dishes into one 50-mL sterile centrifuge tube containing 1.0 mL of FBS (to inhibit further protease action).

14. Centrifuge the cells at 100g for 5 min.

15. Remove and discard the supernatant. Gently resuspend the cell pellet in 15 mL of MHB by repeatedly drawing up and releasing from a pipet (not too vigorously; see Note 11).

16. Prepare 16 mL of 70% FBS in MHB (11.2 mL of FBS plus 4.8 mL of MHB) in a 50-mL of centrifuge tube, and 16 mL of 40% FBS in MHB (6.4 mL of FBS plus 9.6 mL of MHB) in another 50-mL tube.

17. Prepare an FBS gradient in a 50-mL round-bottom centrifuge tube. To do this, carefully pipet 15 mL of the 70% FBS–MHB solution (from step 16) into the bottom of the tube. Very slowly overlay this with 15 mL of the 40% FBS–MHB solution (from step 16), using a pipet held at a 45° angle against the side of the tube just above the 70% FBS layer and slowly releasing the 40% FBS solution in a thin stream so as not to deform the surface of the 70% FBS layer.

18. Let the tube stand undisturbed for 30 min (at room temperature) to allow the larger multinucleated RAW-OCs to settle under normal gravity and penetrate the FBS layers (see Note 12).

19. Carefully pipet off the top 17 mL which contains mononuclear cells, and place into a 50-mL tube.

20. Then, remove the 16-mL middle layer, which contains primarily mononuclear cells and some small multinucleated RAW-OCs, and place into another 50-mL tube.

21. The bottom 12-mL fraction contains predominantly large multinucleated RAW-OCs. RAW Osteoclast Formation 159 22. Centrifuge the purified RAW-OC bottom fraction (and the other fractions if they are also to be cultured and/or analyzed) at 100g for 5 min.

23. Gently resuspend the RAW-OC pellet in culture medium, count an aliquot in a hemocytometer, and plate 1000–4000 cells per well of a 24-well dish. Typically, the purified RAW-OCs from two 100-mm dishes can be cultured in 2–10 wells of a 24-well dish (with 0.5–1.0 mL of medium per well) for 6–24 h (see Note 13). The top and middle fractions from the serum gradient fractionation are typically cultured in 20–40 wells and 15–30 wells of a 24-well dish, respectively. Alternatively, RAW-OCs (and the top and middle fractions, if desired) may be used immediately for analysis (see Subheading 3.2., step 5). Serum gradient fractionation routinely provides 4000–10,000 purified RAW-OCs from one 100-mm dish (this depends on the efficiency of one’s technique and, more importantly, on the exact stage of RAW-OCs used to purify the cells; see Notes 5, 7, 12, and 13). In unfractionated RANKL-generated RAW-OC cultures, multinucleated (more than three nuclei) RAW-OCs typically represent ~1% on a per cell basis and 25% on a per nuclear basis of the total cell population (Fig. 1, left panel). In contrast, serum gradient purified RAW-OCs (more than three nuclei) typically comprise 60–90% on a per cell basis and 96% on a per nuclear basis of the total cell population in the bottom serum fraction (Fig. 1, lower right panel). On average, RAW-OCs in this bottom serum fraction contain 15–30 nuclei per cell.

Alexandr Chernov Thank you Alexandr! That definitely helps! It's a great idea to do the serum gradient.

Interestingly, in most papers, they cultured RAW 264.7 cells on their biomaterials and then did resorption pit assay after specific time-point (21 or 28 days). I'm really wondering how they deal with with the huge number of RAW cells after 21 or 28 days.