{"Equipment":"

\n\tReagents & Buffers

\n
    \n\t
  1. \n\t\tHBSS (Invitrogen14170-138)
    2. \n\t
  2. \n\t\tFetal Calf Serum (FCS)
    3. \n\t
  3. \n\t\tRBC lysis solution (eBiosciences 00-4300-54, made up to 1× with ddH2O)
    4. \n\t
  4. \n\t\tFACS buffer (PBS (-Mg/-Ca), 0.5% FCS, 2 mM EDTA)
    5. \n
\n

\n\tMaterials

\n
    \n\t
  1. \n\t\tForceps & scissors
    2. \n\t
  2. \n\t\tPaper tissues
    3. \n\t
  3. \n\t\t200 μl pipette tips
    4. \n\t
  4. \n\t\t1.7 ml microfuge tubes
    5. \n\t
  5. \n\t\t30 µm CellTrics filters (Partec, 04-0042-2316)
    6. \n\t
  6. \n\t\tDispensing troughs for multichannel pipetting
    7. \n\t
  7. \n\t\t96-well 350 μl polypropylene V-bottom plates (BD Falcon, 353263)
    8. \n
\n

\n\tEquipment

\n
    \n\t
  1. \n\t\tTable top centrifuge
    2. \n
\n","Procedure":"

\n\tSamples are shipped as whole legs in 50 ml tubes containing HBSS on ice from WTSI to KCL (approximately 2 hours by courier) and processed on the same day.

\n
    \n\t
  1. \n\t\tPrepare buffers and antibody master mix (see staining protocol) beforehand. Label plates for staining.
    2. \n\t
  2. \n\t\tPrepare bone marrow extraction tubes – see instructions below protocol.
    3. \n\t
  3. \n\t\tRemove leg from 50 ml tube and remove muscle from the tibia using forceps and scissors. Residual muscle can be removed using paper tissues.
    4. \n\t
  4. \n\t\tPut the leg back into the tube to prevent drying out. Continue until all samples have been prepared up to this step.\n\t\t\n\t
    5. \n\t
  5. \n\t\tOne at time, remove leg from tubes again, and cut at the ends of the tibia using bone scissors. The cut points are just below the knee and just above the ankle. Ensure you can see red marrow through the cross section of the cut.
    6. \n\t
  6. \n\t\tPlace the rest of the leg back into the 50ml tube for contingency in case insufficient marrow is obtained from the tibia.
    7. \n\t
  7. \n\t\tInsert the bone into the bone marrow extraction tube with the widest end on the bone at the bottom.
    8. \n\t
  8. \n\t\tSix at a time, place microfuge tubes into microfuge and spin at 800×g for 30 seconds.
    9. \n\t
  9. \n\t\tInspect tubes to ensure bone marrow has been extracted. Otherwise cut the end off the bone to increase the opening and repeat. If no bone marrow can be extracted, repeat protocol with femur after all other samples have been processed.
    10. \n\t
  10. \n\t\tDiscard the pipette tips containing the bone from the microfuge.
    11. \n\t
  11. \n\t\tResuspend the bone marrow pellet in 50 µl of 1× RBC lysis buffer at room temperature. Pipette several times to break up pellet.
    12. \n\t
  12. \n\t\tIncubate for 1 minute at room temperature.
    13. \n\t
  13. \n\t\tAdd 200 µl FACS buffer.\n\t\t\n\t
    14. \n\t
  14. \n\t\tCentrifuge at 500×g for 30 seconds.
    15. \n\t
  15. \n\t\tRemove supernatant and resuspend in 200 μl FACS buffer.
    16. \n\t
  16. \n\t\tRepeat steps 4-14, six samples at a time, until all the bones have been processed
    17. \n\t
  17. \n\t\tFilter cells into 1.7ml microfuge tubes using 30 µm filters. Rinse tube and filter using 200 µl FACS buffer.
    18. \n\t
  18. \n\t\tPipette 200 µl of each sample into a 96-well plate.
    19. \n\t
  19. \n\t\tCentrifuge plate at 800×g for 1 minute at 8o C.
    20. \n\t
  20. \n\t\tCells are now in single cell suspension on plates and ready for staining (see staining protocol).
    21. \n
\n

\n\tPreparation of bone marrow extraction tubes

\n
    \n\t
  1. \n\t\tCut a 200 ml pipette tip at the 10 μl line and just below the line where the tip of the pipette would end (see picture, colour for illustration only).
    2. \n\t
  2. \n\t\tInsert the middle section of the cut tip into the top section
    3. \n\t
  3. \n\t\tPlace into a 1.7 ml tube.
    4. \n
\n

\n\t 

\n","Purpose":"

\n\tProtocol for isolation of bone marrow and processing into single cell suspension

\n"}