Endosteal bone marrow Td+ cell isolation and cell sorting

1. harvest of mice hind long bones

1) Euthanize Col2-tomato mouse

2) Using sterile forceps and scissors, incise and peel back the skin surrounding the hind long bones. Remove the bilateral hind long bones by cutting through the hip and ankle joints using sharp surgical scissors. Cut through the knee joint to separate the tibia and femur

3)  Place the long bones in a 100 mm petri dish. Transfer the petri dish with the bones to a tissue culture hood for bone marrow harvesting

2. Isolation of endosteal bone marrow cells

1) Under a sterile tissue culture hood, use forceps and a scalpel to remove all of the soft tissue surrounding the bones.

 2) Cut off both ends of the tibia and femur at the growth plate with a scalpel

3) Fill a syringe with 5 ml of flushing medium per mice. Attach a 27G needle to the syringe and then place the filled syringe on the side of the workspace

4) With the prefilled syringe, place the needle in the hole at one end of the bone and press down the plunger to force medium through the bone. This will flush the bone marrow out of the bone through the opposite end of the bone.

5) Reverse the bone and repeat the flushing from the other end of the bone. Use 1ml of flushing medium to flush out each bone. 

7) After removing the central bone marrow cells, scrape the outside surface of the bones a few times with a scalpel blade and then place the bones into a 15 ml tube containing 5ml of protease solution (2mg/ml collagenase A, Roche diagnostics, Indianapolis, IN; and 2.5 mg/ml trypsin dissolved in Dulbecco’s PBS and filter sterilized using a 0.2μm syringe filter).

8) Place the tubes on a mini shaker placed in the tissue culture incubator and shake for 20 min at 37°C. 

9) After digestion, wash the bones with flushing medium, and then longitudinally cut the bones into two halves

10)Use a syringe to gently wash the inside of the bones with flushing medium to remove loosely attached bone marrow

11)  Place the bone fragments into a 15ml tube containing 5ml of protease solution and perform the second digestion step for 60 min

12)  Collect the supernatant and then add 5ml of growth medium (α-MEM supplemented with 15% FBS, 0.1% β mercaptoethanol, 20 mM glutamine, 100 IU/ml penicillin and 100ug/ml streptomycin) to neutralize the protease solution

13)  Centrifuge at 300Xg for 5min to pellet the cells

14)  Resuspend the cells in 5ml of growth medium by gently pipetting up and down several times and then centrifuge.

15)  Resuspend the cells in 3ml of growth medium and then pass the cells through a 70 μm cell strainer to remove debris.

16)  Perform a cell count by diluting a cell aliquot 1:10 in 3% acetic acid with methylene blue to lyse the red blood cells

17)  Count the nucleated cells using a hemocytometer under a microscope. 

Prepare:

10 ml sorting medium: 9.55ml DPBS+200ul FBS+250ul HEPES (final concentration is 25mM )

10 ml collection medium: 9.75 ml growth medium+250ul HEPES, 1ml per collection tube

18)  Cells for FACS were centrifuged and then resuspend into sorting medium in polypropylene FACS tubes (Falcon cat# 352063) 10million/ml sorting medium. Keep it on ice, use Influx B sorter to sort tomato+ cells. 

21) Sort cells with low speed at 1500 events/second and low pressure setting with 140um large nozzle. Collect top 1% tomato+ cells for single cell RNA sequence.

After sorting, take some cells to check cell viability. Add 0.5 ul DAPI to 5 ml sorted cells. Quickly run sorting. 97% cells are live cells.

22) Bring sorted tomato+ cells to single cell sequence core. 

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