BMSC isolation, cell culture, and immunostaining

I have attached our detailed step by step protocol.  It is quite challenging to do this and I would recommend having at least two people helping to make sure the dissection and marrow isolation from the mice goes as quickly as possible.  You will also need to pool bones from 9 to 12 mice per marrow fat sample.  So, this experiment requires a lot of mice.  You can do less if you use a larger species such as rats (ex. 2 rats per sample). If you would like to meet briefly to discuss the details, please feel free to email me directly (scheller@wustl.edu).

Bone marrow adipocyte purification and RNA extraction - mouse

Scheller Lab

Buffer #1 (50 mL): HBSS (with calcium and magnesium, Gibco 10425-076) + 2% DNase and protease free BSA (Fisher BP9706) + 5 mM EDTA (diluted from the 14% EDTA, pH 7.4 stock – 520 uL stock into 50 mL of HBSS) + 1 g/L glucose

Buffer #2: 10 mL Buffer #1 + 1 mg/mL collagenase 

Notes:

• 1 g/L glucose was added to provide a substrate for the adipocytes and promote survival
• Recommend pooling 9 to 12 mice per prep, this will yield ~100-500 ng of total RNA if there are a reasonable amount of starting BMAT adipocytes (ex. older B6 mice or C3H mice)
• Move quickly!  Get help from 1 to 2 additional people for the dissection and initial cell isolation if possible.
• We were able to do one prep per half day (ex. one in the morning and one in the afternoon)
• The ultimate goal is to get as pure of a BMAT population as possible, though we would still call this “bone marrow adipocyte enriched” (BMAe)
• Use known adipocytes vs bone vs bone marrow target genes to validate the quality of your preps (ex. Pparg, Alpl)

Filter columns and RNA/gDNA combo isolation kits:

• Exon Biosciences filter columns (possible alternatives Pierce 69700 and 69725)
• Nucleospin RNA/DNA Buffer Set (Macherey-Nagel 740944)
• Nucleospin XS (Macherey-Nagel 740902.50)

Protocol: 

1. Euthanize with CO2 
2. Spray with 70% ethanol to prevent fur from going everywhere 
3. Remove and clean femurs and tibiae – put into a 10 cm plate with room temperature buffer  
   1. Femur 
      1. Remove the distal epiphysis  
      2. Cut the proximal end to expose the marrow cavity 
   2. Tibia 
      1. Remove the proximal epiphysis 
      2. Cut at the level of the tibia/fibula junction to expose the marrow cavity 
4. Flush bone marrow using a 10 mL syringe + 22 ga needle into a 50 mL conical tube (20 mL total buffer) – Tube “BMAT #1a”
   1. Puncture the metaphysis near the knee and use the liquid to push out the marrow plug 
   2. Try to rinse the metaphysis to remove remaining marrow
   3. Every ~4 bones, draw the marrow plugs from the bottom of the tube through the needle to disperse them 
   4. Refill the syringe as needed by putting the needle to the bottom of the tube and drawing up new liquid 
   5. Put the bone fragments (end caps and diaphysis) into a second tube (Tube BMAT #1b) containing Buffer #2
      1. After putting ~half of the bones in, mince them into fine fragments to allow the collagenase to liberate any remaining adipocytes from the bones/marrow
      2. Re-mince periodically until the end of the flush
5. Centrifuge tubes BMAT 1a and 1b at 400 g for 2 minutes to pellet the bone marrow and float the adipocytes 
6. Decant the supernatant from BMAT 1a and 1b tubes + the adipocytes into a new 50 mL tube without removing the red pellet or bone fragments – Tube “BMAT #2”
   1. Centrifuge at 400 g for 2 minutes to pellet any remaining marrow and float the adipocytes 
   2. Remove the infranatant and pellet with a pulled glass pipet until only ~ 10 mL of liquid remains 
7. Decant the supernatant + the adipocytes into a new 15 mL tube without removing any residual red pellet fragments – Tube “BMAT #3”
   1. Centrifuge at 400 g for 1 minute to pellet any remaining marrow and float the adipocytes 
   2. Remove the infranatant and pellet with a pulled glass pipet until only ~1 to 2 mL of liquid remains 
   3. **Put a small sample of this onto a glass slide with trypan blue to confirm the presence of viable adipocytes and the depletion of hematopoietic cells**
8. Resuspend the adipocytes into the remaining liquid and put into a filter column (see info and images on Page 1)
   1. Slowly centrifuge the column at 50 rcf for 10 seconds to remove any liquid 
   2. Repeat with the remaining volume until depleted
   3. When liquid is depleted, proceed to cell digestion ON THE COLUMN for RNA isolation (Sample = BMAT RNA)
      1. Put parafilm over the hole of the column to block the lysis buffer from escaping
      2. Load lysis buffer onto the filter column, pipet 3X 
      3. Incubate for 3 minutes at RT with gentle ‘flicking’ to agitate the sample
      4. Spin at 11,000 rcf for 30 sec
      5. Re-load the same buffer onto the column, re-spin as above
      6. Re-load the same buffer again onto the column, allow gravity to pull the liquid through
         1. Sometimes won’t go through with gravity so do extra 30 sec, 11,000 rcf spin
      7. When empty, spin once more
      8. Continue with RNA isolation kit using the elutate (BMAT RNA lysate)