The overall goal of this procedure is to isolate and label neutrophils from mouse bone marrow for downstream functional studies and adoptive transfer experiments. This is accomplished by first collecting the bone marrow cells from murine, femurs and tibia. During the second step, the red blood cells are osmotically laced by sequentially suspending them in 0.2%and 1.6%sodium chloride solutions respectively.
Next, the neutrophils are isolated by density gradient centrifugation. In the final step, the neutrophils are labeled with cell tracker dyes. Ultimately, flow cytometry can be used to determine the number of adoptively transferred neutrophils recruited to various mouse tissues.
The main advantage of isolating mouse neutrophils from the bone marrow over blood or the peritoneum, is that the large number of cells sufficient for downstream functional studies and adoptive transfer experiments can be obtained from the bone marrow both during the steady state and after infection. The advantage of using a gradient centrifugation technique for isolating mouse neutrophils over immuno magnetic selection of fluorescence activated cell sorting is that it is fast economical and it avoids labeling the neutrophils with antibodies which may induce neutrophil activation. Demonstrating the procedure will be muca swami us, a technician from my laboratory After spraying down a euthanized mouse with 70%ethanol.
Make an incision in the mid abdominal region of the animal's skin and then remove the skin tissue from the distal part of the animal, including the lower extremities. Next, use scissors to cut the muscles from the animal's hind limbs and then carefully dislocate the ACE beum from the hip joint, avoiding breaking the head of the femur. Then use the scissors in a scalpel to remove the remaining muscle from the femur and tibia.
Separate the leg bones at the knee joint, taking care not to break the bone ends, and then place the bones in a Petri dish containing ice cold RPMI 1 6 4 oh. Supplement with FBS and antibiotics. Transfer the dish to a tissue culture hood and then sterilize the outside of each bone with 70%ethanol in a new Petri dish.
After about 10 to 15 seconds, rinse the ethanol from the surface of each bone with three subsequent washes in ice cold, sterile PBS. Then cut off and place the epiphysis from each of the bones into a new sterile Petri dish. Next, attach a 25 gauge needle to a 12 cc syringe filled with 10 milliliters of RPMI.
Supplement with FBS and EDTA. Then flush the bone marrow cells from both ends of the shaft of each femur tibia pair onto a 50 milliliter screw top Falcon tube faded with a 100 micrometer filter to remove all of the cells. Scrape the inner surfaces of the bones with the needle tip.
When the bones appear blanched, the cells have been sufficiently removed. Now use a scalpel to cut the bone epiphyses into small 0.5 to one millimeter Q pieces, and then use the back end of a 2.5 milliliter einor comb tip plus BioPure pipette tip to smash them through the 100 micrometer filter. After spinning down the harvested cells for seven minutes at 427 times G and four degrees Celsius ly the red blood cells in 20 milliliters of 0.2%sodium chloride for approximately 20 seconds.
Then stop the lysis with 20 milliliters of 1.6%sodium chloride, spin down the cells and then resuspend the pellet in RPMI 1 6 4 oh supplement with FBS and EDTA. Count the cells and then after spinning them down again, resuspend the washed pellet at no more than 100 times 10 to the six cells per one milliliter of ice. Cold sterile PBS Now dispense three milliliters of 18 to 26 degree Celsius, his opaque 1 1 1 9 into a 15 milliliter conical tube, and then slowly overlay three milliliters of 18 to 26 degree Celsius.
His opaque 1 0 7 7. Avoiding mixing the two densities immediately after preparing the his opaque carefully layer the bone marrow cell suspension on top of the his opaque 1 0 7 7 centrifuge, the density gradient for 30 minutes at 834 times G and 25 degree Celsius without the break, and then collect the neutrophils at the interface of the his opaque 1 1 1 9 and 1 0 7 7 layers. After washing the collected neutrophils twice in RPMI 1 6 4 oh FBS and antibiotics Resus suspend the cells at five times 10 to the six cells per milliliter in PBS Prewarm to 37 degrees Celsius.
After counting the cells, reserve a small aliquot of each experimental sample to determine the purity and viability by flow cytometry and then label the remaining neutrophils with a cell tracker dye at a final concentration of five micromolar for 10 minutes at 37 degrees Celsius in a shaking water bath in the dark. Finally, wash the cells twice with ice cold RPMI 1 6 4 oh supplement with FBS and antibiotics to avoid dye cross-contamination before mixing the differentially labeled neutrophil populations for downstream competitive repopulation studies here, representative flow cytometry plots of bone marrow isolated density gradient separated neutrophils from an uninfected C 57 black six mouse are shown in this panel. The initial gating used for the bone marrow cells collected from the interface of histopath 1 1 1 9 and histopath 1 0 7 7.
Using forward and side scatter is demonstrated the density gradient separated neutrophils are greater than 90%pure and greater than 95%viable for at least four hours after adoptive transfer cell tracker. DIA label neutrophils may be tracked by flow cytometry in various mouse issues. For example, in the blood bone marrow and kidneys of the recipient candida infected C 57 black six mouse shown in this experiment by gating on the live CD 45 LY six G CD 11 B cells, these representative adoptively transferred C-M-T-M-R labeled neutrophils can be differentiated from the native C-M-T-M-R and PE negative neutrophils of the recipient mouse by their PE expression.
Once mastered the isolation and labeling of the mouse bone marrow neutrophils can be completed in three hours.
