Hello!

I use 50ml DMEM 10% FBS supplemented with 25ng/ml MCSF (Peprotech) to re-suspend bone marrow obtained from flushing both femur and tibia of mouse.

After that, I leave the progenitors in culture for 7 days w/o changing the medium or adding more supplements. During the differentiation the cells will start to produce their own growth factors and will grow happily.

As long as your bone marrow suspension has been filtered properly with a cell strainer you shouldn't have lots of debris and so the culture be "clean".

Hope this helps,

Fede

Hi Sreeram Udayan,

I thinking you mean generation  of bone marrow-derived macrophages. The reference is my own paper.  http://www.sciencedirect.com/science/article/pii/S017193351400106X

1. Prepare the conditioned Medium :

DMEM Gibco 21969 = 50ml

50μM ME Sigma Aldrich M-7154= 0.25ml

1% nonessential amino acids Gibco 11140 = 0.5ml

10%fetal calf serum PAA A15-10= 2 5ml

Horse serum=2.5ml

Hepes (1M) Gibco 15630 = 0.5ml

L-Glutamin (200mM) Biochrom AG K0282 =1ml

L929- Supernatant =7.5ml

2. Scarify the mouse by cervical dislocation or CO2.

3. Spray all external areas of the mouse with 70% ethanol and fixing the mice to the post-mortem board with clean absorbent paper.

4. Using a blunt end scissor, make an incision 1 inch vertically from umbilical region to anterior region.

5. Extend this incision along the medial aspect of both rear appendages and gently pull the skin downward below the heels to expose the muscles, etc.

6. Using a sharp scissor, dissect tibia and femur from surrounding muscles and tendons and place the tibias and femurs into a 50 mL polypropylene tube containing DPBS.

7. Using a sharp scissor, remove excess tissue, the epiphysis of the knee and heel as smaller as possible in the conditioned Medium to open the bone marrow.

8. Fill the 1ml syringe (with26G needle) with conditioned Medium and insert into the opened bone marrow cavity. And then flush the BM with the Medium.

9. Collect the stem cells into 50ml falcon tube and centrifuge at 1500rpm, 10 minutes, 4° C.

10. Discard the supernatant carefully, add 3ml medium to the sediment and re-suspend.

11. Count the cells under microscopy after mixing with Turk`s solution in 1:20 ratio (cell to Turk`s solution).

12. Determine the number cells and amount of Medium you need.

13. Add 10ml of the cells in conditioned Medium to plate(s) and incubate at 37 ° C for at least 6 days.

14. Harvest bone marrow derived macrophages (BMDM) and work with it.

Thank you Rebuma Firdessa. Yes, you're right, I meant bone-marrow derived macrophages, but I want to generate them using recombinant MCSF (not L929), everything else the same. But thanks very much for the reference.

Hi Sreeram Udayan again,

L929 supernatant contains MCSF  that we produce from L929 cell line. So, is it not simply using recombinant MCSF in place of L929 supernatant? The dose supposed to be used may vary.

I treat BMDM with 10ng/ml MCSF (http://www.peprotech.com/en-US/Pages/Product/Recombinant-Proteins/Growth-Factors-Cytokines/Recombinant_Murine_M-CSF/315-02) every 2-3 days after isolation for 6 days.

Before switching to L929 conditioned medium, we used 10 ng/ ml M-CSF to generate bone marrow-derived macrophages (one medium replacement event, after 3-4 days; total culture time 6-7 days). The following reference may be helpful:

Autophagy regulates phagocytosis by modulating the expression of scavenger receptors.

Bonilla DL et al

Immunity. 2013

PMID: 24035364

Thanks Maria, Thanks Abhisek

Hello

For reparation of L929-Conditioned Medium :

1. Plate 4.7 × 105 L929 cells in a 75-cm2 flask containing 55 mL of L929 medium.

2. Grow cells in a humidified incubator with 5% CO2 at 37°C for 7 d.

3. Collect the supernatant. Filter through a 0.45-μm filter. Store 50-mL aliquots frozen at −20°C (L929-conditioned medium).

the protocol you can use :

Preparation of L-cell conditioned medium: culture L929 cells with initial 50% confluence in RPMI + 10% FBS for 5 days, collect the medium and filter with 0.22 μm filter, aliquot and store at -20 °C.

Note: Incubate FBS at 50 °C for 30 min before using.

Prepare bone marrow growth medium and BMM’phi’ growth medium (see Recipes).

Isolation of mouse bone marrow cells.

Sacrifice mouse and immerse mouse in 75% ethanol.

Clip the skin mid-back and remove the skin from the lower part of the body.

Remove tissue from legs with scissors and dissect away from body.

Clean remaining tissue from the pelvic and femoral bones and separate at knee joint. Be careful not to break the bones. It is important to make sure that all the tissue is removed from the bones since cells associated with this can contaminate the marrow preparation and potentially overgrow the macrophages.

Immerse the bones in 75% ethanol for 5 min, then immerse them in DPBS for 5 min and leave them in RPMI+P/S until the next step.

Cut off each end of bone.

Using a 27 g needle/1 ml syringe filled with bone marrow growth medium; expel the bone marrow from both ends of the bone with a jet of medium directed into a 15 ml cell culture dish.

Change medium every 3 days. While in culture, some of the cells become attached, while many of them still grow in suspension, so spin-down and re-culture those cells in new dishes.

After about 10 days, almost all cells become attached BMM’phi’s, and then BMM’phi’ growth medium is used for further culture and tests.

Also , you can see this links .

Good Luck

http://cshprotocols.cshlp.org/content/2008/12/pdb.prot5080.full.pdf+html

http://www.cellandbioscience.com/content/pdf/2045-3701-3-30.pdf

Hello!

I use 50ml DMEM 10% FBS supplemented with 25ng/ml MCSF (Peprotech) to re-suspend bone marrow obtained from flushing both femur and tibia of mouse.

After that, I leave the progenitors in culture for 7 days w/o changing the medium or adding more supplements. During the differentiation the cells will start to produce their own growth factors and will grow happily.

As long as your bone marrow suspension has been filtered properly with a cell strainer you shouldn't have lots of debris and so the culture be "clean".

Hope this helps,

Fede

Thanks Saleh Al Karim

Thanks Federica, It's just what I wanted to know :-). My MCSF is also from Peprotech. If you could tell me what is your preferred storage buffer for MCSF, that would be great. Thanks. 

Yes of course. I make it up in water first to a higher concentration and then I diluite it down with PBS+0.1% BSA before aliquoting. I store the aliquotes at -80C.

Hope this helps,

Fede

Hi Fede, 

Do you think it would be okay if my DMEM contains sodium pyruvate?

Also how much L-Glutamine do you supplement your media with?

Thank you.

Kind Regards,

Mohit