A stepwise procedure for isolation of murine bone marrow and generation of dendritic cells
2 Department of Biotechnology, Jamia Hamdard, New Delhi-110062, India
Keywords: bone marrow, dendritic cells, primary culture
- RPMI-1640 medium (Life technologies, India)
- Fetal Bovine Serum FBS (Life technologies, India)
- Penicillin-streptomycin (Himedia, India)
- Ethanol (Sigma, USA)
- HBSS (Sigma, USA)
- Trypan blue (Sigma, USA)
- Recombinant mouse granulocyte-macrophage colony stimulating factor (rmGM-CSF), 1000 ng/ml (R&D systems, Minneapolis, USA)
- Lipopolysaccharide (LPS, from E. coli serotype 0127:B8)- 100 ng/ml (Sigma, USA)
- FITC-conjugated CD11c (HL3) (557400, BD Biosciences PharMingen, USA)
- DMSO (Merck, India)
- Disposable syringe (1 ml Insulin) 29G × ½ (Dispovan, U-40)
- Kleenex tissues
- Petridish (Non-TC grade)
- Pipette tips
- 15 ml and 50 ml centrifuge tubes
- Centrifuge (Heraeus)
- CO2 Incubator (Shell labs)
- Hemocytometer (Sigma, Z37,535-7)
- Inverted microscope (Nikon Diaphot-300)
- Laminar air flow (Klenzaids)
Isolation of bone marrow – day 0
1.Euthanize a C57BL/6 mouse (male, 20 to 25 g, 8–10 weeks old) by CO2 asphyxiation. Subsequent experimental steps will be conducted in a laminar air flow.
2.Position the mouse on its dorsal back on a clean blotting sheet and thoroughly spray all the external areas with 70% ethanol for disinfection. This step minimizes the possibility of contaminating cell preparations with fur or external particles.
3.Make an incision in each hind leg using blunt-end sterile scissors. Firmly grasp the skin and gently pull downwards to expose the muscles.
4.Cut the hind leg just above the pelvic/hip joint using sharp and sterile dissecting scissors, ensuring that the epiphysis remains intact without exposing off its contents to outside.
5.After removal of the hind leg, carefully hold it from the lower side. Using sterile scissors make an incision just above the claws to remove the lower portion of the hind leg.
6.Transfer the hind leg to RPMI-1640 medium in a sterile petridish and keep for 5 min to loosen the muscle tissue.
7.Cut the hind leg at just below the knee-joint through ligaments to remove off the tibia, ensuring that the epiphysis remains intact.
8.Dissect the femur from surrounding muscles and remove excess tissue using sterile forceps and scissors, keeping the ends of the bone intact.
9.Remove any extra leftover muscle/tissue on the femur by using lint-free tissue paper (Kleenex) and gently clean the bones using 70% ethanol.
10.Soak the intact bones in 70% ethanol in a sterile petridish for 2 min for disinfection. Repeat the same procedure with another femur.
11.Transfer the bones to HBSS in a sterile petridish for rinsing off ethanol.
12.After rinsing, transfer the bones to culture medium RPMI-1640 in a sterile petridish.
13.Trim both ends of femurs carefully using sterile, sharp scissors to expose the interior marrow shaft.
14.Flush the contents of marrow with 2 ml of HBSS using a 1-ml insulin syringe with a 29G × ½ needle. Collect the contents into a sterile 50-ml centrifuge tube. (The bones should appear white once all the marrow has been expelled out completely).
Preparation of BM cell suspension – day 0
15.Dilute the BM cell suspension collected above with HBSS to a final volume of 20 ml. Disintegrate any clusters within the bone marrow suspension by vigorous pipeting.
16.Centrifuge the cell suspension at 250 g × 8 min.
17.Remove the supernatant and resuspend cell pellet in 20 ml of HBSS. Centrifuge the cell suspension at 250 g × 8 min (wash 1).
18.Again wash the cell pellet in HBSS as mentioned above (wash 2).
19.After 2 washes, resuspend the cell pellet from each femur gently in 20 ml of HBSS to prepare a homogeneous suspension. Take out an aliquot of cell-suspension for cell count.
20.Count the total number of cells using a hemocytometer and trypan blue staining method for cell viability.
21.Centrifuge the cell suspension at 250 g × 8 min.
22.According to the cell count obtained, resuspended the cell pellet in culture medium (RPMI-1640 + 10% FBS + 20 mM penicillin/streptomycin) to achieve a final cell density of 10 × 106 cells/ml.
Seeding of cells (day 0)
23.Add 9.6 ml of culture medium (RPMI-1640 + 10% FBS + 20 mM penicillin/streptomycin) into sterile, pre-labeled 90-mm petridishes (non-tissue culture grade).
24.Add 0.2 ml of cell suspension (10 × 106 cells/ml) into each petridish to achieve the final cell density of 2 × 106 cells/petridish.
25.Add 0.2 ml of rmGM-CSF from rmGM-CSF stocks (1000 ng/ml) into above petridishes so that final concentration of rmGM-CSF in 10 ml is 20 ng/ml.
26.Gently swirl the petridishes to ensure uniform mixing of contents.
27.Incubate the cells at 37°C, 5% CO2 and 95% humidity in CO2 incubator for 3 days.
Replenishment of culture with fresh culture medium (day 3)
28.Add 10 ml of fresh culture medium with 20 ng/ml rmGM-CSF into each petridish with BMDC cultures. The total volume in each petridish is now 20 ml.
29.Gently rotate the petridishes for proper mixing of contents.
30.Incubate the cultures at 37°C, 5% CO2 and 95% humidity in a CO2 incubator further for another 3 days.
Harvesting of BMDCs (day 6)
31.Harvest primary BMDCs from each petridish by collecting non-adherent cells by gently pipeting them with culture medium. Loosely-adherent BMDCs get easily dislodged into suspension by this process while the adherent
macrophages remain attached to the petridish.
32.Collect the cell suspension from each petridish into sterile 50-ml
centrifuge tubes. Discard the adherent cells which contain macrophages.
33.Transfer the cell suspensions to polypropylene tubes for FACS analysis.
34.For treatments in biological assays, centrifuge the cell suspension at 250 g × 8 min.
35.Resuspend the cell pellet in culture medium (RPMI-1640 + 10% FBS + 20 mM penicillin/streptomycin) to make a homogeneous suspension.
36.Count the cells using a hemocytometer and trypan blue staining method for cell viability.
37.Adjust the cell density as per required number for biological assay.
Flow cytometry analysis of BMDCs
38.Harvest day-6 immature BMDCs from 90-mm petridishes.
39.Count the cells and collect 1 × 106 cells into each sterile centrifuge tubes.
40.Centrifuge the cells at 250 g × 8 min.
41.Remove the supernatant and resuspend the cell pellet in 5 ml of PBS.
42.Centrifuge the cells at 250 g × 8 min (wash 1).
43.Remove the supernatant and repeat the wash once (wash 2).
44.Incubate the cells with FITC-labeled CD11c antibody and isotype control in 100 µl of PBS, at 4°C in dark.
45.After staining for 30 minutes, add 0.9 ml of PBS into each tube.
46.Repeat the wash steps twice by centrifuging the cells at 250 g × 8 min.
47.Remove the supernatant and resuspend the cell pellet in 0.3 ml of buffer (PBS + 5% FBS).
48.Transfer the cell suspensions to polypropylene tubes for FACS analysis.
1.On day 0, the expected bone marrow yield is 10 ± 1.654 × 106 (per femur) (n = 25).
2.On day 6, the expected BMDC yield is 2 ± 0.661 × 106 (per 90-mm dish) (n=25).
Morphology of BMDCs
3.On day 0, bone marrow progenitor cells seeded into culture petridishes show spherical morphology (Fig. 4A). Cells are small in size but with a defined cell-membrane and good health.
4.On day 3, BMDCs demonstrate spherical morphology with very early stages of “dendrites” formation on their cell surface. Initiation of colony formation can be seen at various sites with aggregation of cells together (Fig. 4B). Conversion of cells into adhered macrophages is also seen (elongated cells).
5.Immature BMDCs are obtained on day 6 of the culture in the presence of rmGM-CSF. At this stage, differentiation of cells into adherent macrophages is observed. Simultaneously, large sized colonies of BMDCs are formed at various sites (Fig. 4C). Large numbers of semi-adherent and floating BMDCs are observed.
6.Treatment of immature BMDCs on day 6 of culture with maturation stimulus LPS (100 ng/ml) for 24 h results in the maturation of BMDCs as evident by an increased population of cells with branched and extended morphology and adhered macrophages (Fig. 4D).
Purity of BMDCs
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