BMDC isolation protocol
This protocol will guide you through isolating immature bone marrow-derived dendritic cells (BMDCs) from mice.
Stage 1 - Isolation
Ensure that all the reagents and samples are kept on ice from steps 1–6 as this has a positive impact on cell viability.
In order to maintain sterility, perform all steps in a laminar flow hood with sterile utensils. Watch our video guide on aseptic technique.
Steps
Cut the back legs above the hip joint
- Cut the back legs above the hip joint in order to access the femur and tibia, leaving knee and ankle joints in place.
- Debride the muscle and tissue from by rubbing with KimwipesTM.
The femur tends to have more marrow than the tibia.
Use only intact bones, as the bone marrow will not be sterile if they break.
Fill a petri dish with 70% ethanol.
- Fill a petri dish with 70% ethanol and dip the cleaned bones into it for 5–10 seconds to sterilize their exteriors before keeping them in a sterile tube on ice until all the bones are flushed.
In a laminar flow hood using sterile utensils, cut both ends of the bone with scissors as close to the joints as possible.
- Fill a syringe with ice-cold RPMI complete (R10) media, insert the syringe needle into the bone and flush out the bone marrow into a centrifuge tube on ice.
- Flush 2-3 times until the bones are completely white.
- Dissolve any clusters by pipetting.
Centrifuge 1-2 times in R10 media at 300 x g for 5 minutes.
Resuspend the cell pellet in R10 and count viable cells using a hemocytometer and trypan blue.
Dilute the cells into 10 mL of R10 + 20 ng/mL GM-CSF.
- Plate in petri dishes at a density of 2 x 106 viable cells per plate.
Place plates in a 37°C incubator with 5% carbon dioxide.
At day 3, add an additional 10 mL of R10 + 20 ng/mL GM-CSF.
At day 6, remove half of the media (10mL).
Briefly centrifuge the removed volume of media.
- Resuspend in 10 mL of fresh R10 + 20 ng/mL GM-CSFand add this back into the original culture.
Cells can be harvested on day 8, or step 10 can be repeated to harvest cells on day 9 or 10.
Cells should be only briefly centrifuged as the DCs are only loosely adherent.
Non-adherent and loosely adherent cells in the culture in the culture supernatant can be harvested by gentle washing with PBS, and then pooled for subsequent experiments.
The majority of adherent cells will be macrophages, while those that are in suspension or loosely adherent will be dendritic cells (DCs) and F4/80+ macrophages. For a highly pure fraction, it is recommended to take measures to deplete F4/80+ cells and enrich the population for CD11c+ cells.
Most of the macrophages in suspension will dually express F4/80+ and CD11c. The F4/80+ cells can be removed via positive selection with anti-F4/80 magnetic beads. After this step, CD11c+ DCs can be positively selected from the flow-through using anti-CD11c magnetic beads.
The immature DC’s can be used immediately after purification, or further cultured if the user wishes to obtain mature cells.
Purity assessment
- It is recommended to perform flow cytometry on an aliquot of cells to check for purity. A purity check reagent can also be included to label cells still bound to magnetic beads.
Suggested markers
Anti-CD11c antibody [N418] (ab33483). This is also available labeled with Biotin, FITC, APC/Cy7, PE, PE/Cy7, PerCP/Cy5.5 and as low-endotoxin, azide-free.
Anti-CD11b antibody [EPR19373] (ab184307)
Anti-F4/80 antibody [BM8] (ab16911) or Anti-F4/80 antibody [CI:A3-1] (ab6640). These antibodies are also available directly conjugated to FITC, PE or APC.