BMDC Isolation Method
Licia Miller Product Manager
The isolation and culture of BMDC (bone marrow-derived dendritic cells) is an important technique in immunology research. This protocol will introduce how to isolate immature bone marrow-derived dendritic cells from mice.
Procedure
Make sure all reagents and samples in steps 1-6 are kept on ice to maximize cell viability.
To maintain sterility, perform all steps in a laminar flow hood using sterile instruments.
1. Cut the hind leg above the hip joint to access the femur and tibia, leaving the knee and ankle joints intact. Use KimwipesTM to clean the muscle and tissue scars. The femur generally has more bone marrow than the tibia.
NOTE: Only whole bones should be used, as the marrow will not be sterile if the bone is broken.
2. Pour 70% ethanol in a petri dish and immerse the cleaned bones in it for 5-10 seconds to sterilize the outside of them, then place them in a sterile tube on ice until all the bones have been rinsed clean.
NOTE: You can leave the bones in the ethanol longer as you rinse each bone, but be careful not to soak them too long , as the ethanol will soak into the bones and kill living cells.
3. Using sterile instruments in a laminar flow hood, cut the two ends of the bone with scissors , paying attention to being as close to the joint as possible. Then fill a syringe with ice-cold RPMI complete medium (R10), insert the syringe needle into the bone and flush the bone marrow into a centrifuge tube placed on ice.
4. Rinse 2-3 times until the bones are completely white. Dissolve all the clusters by pipetting.
5. Centrifuge 1 - 2 times at 1,500 rpm for 5 minutes each in R10 medium.
6. Resuspend the cell pellet in R10 medium and count the viable cells using a hemacytometer and trypan blue staining.
7. Dilute the cells into 10 mL R10 + 20 ng/mL GM-CSF. Then transfer the cells to culture dishes at a density of 2 × 106 viable cells per plate.
8. Place the culture dish in a 37°C incubator containing 5% carbon dioxide.
9. On day 3, add another 10 mL of R10 + 20 ng/mL GM-CSF.
NOTE: Add media very gently to avoid disturbing growing cells.
10. On day 6, remove half of the culture medium (10 ml).
NOTE: Remove medium very gently to avoid disturbing growing cells.
11. Remove the culture medium and centrifuge briefly. Resuspend the cell pellet in 10 mL of fresh R10 + 20 ng/mL GM-CSF and add it back to the original culture.
Note: Cells can be harvested on day 8, or step 11 can be repeated to harvest cells on day 9 or 10.
Since dendritic cells (DCs) are poorly adherent, the cells should only be centrifuged briefly.
12. Non-adherent cells and loosely adherent cells in the culture supernatant can be gently washed with PBS and then pooled for subsequent experiments.
Warning: Avoid using EDTA here, as this will remove adherent macrophages and dilute your DC sample.
Tip: Most adherent cells are macrophages, while suspended or loosely adherent cells are dendritic cells (DC) and F4/80+ macrophages. To obtain a high purity fraction, it is recommended to take steps to deplete F4/80+ cells and enrich the CD11c+ cell population.
Most macrophages in the suspension will express both F4/80+ and CD11c. F4/80+ cells can be removed by positive selection with anti-F4/80 beads. Following this step, CD11c+ DCs can be positively selected from the effluent using anti-CD11c beads.
13. Immature DCs can be used immediately after purification or further cultured if you wish to obtain mature cells.
Tip: DCs have a loose adhesion and only need to adhere to the culture dish to mature, so you can use a 96-well U-bottom plate that has not been treated for tissue culture to prevent premature maturation.
14. Purity Assessment. It is recommended to check the purity of the cells by flow cytometry. A purity check reagent can also be added to mark the cells that are still bound to the beads.
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