Materials for isolation*

  1. Peristaltic pump capable of 1-10mL/minute
  2. Reservoir capable of holding between 50-100mL of solution
  3. Bubble trap
  4. Waterbath @37C
  5. Sterilized 100mL wide-mouth glass bottles
  6. Sterile 50mL conical tubes
  7. 70 micron filter; disposable mesh or reusable stainless steel [1]
  8. 10cm petri/cell culture dishes
  9. Sterilized dissection tools; at minimum, one pair of scissors and two pairs of fine-tip forceps
  10. 70-75% EtOH and detergent, both in spray bottles (iodine optional)
  11. Anesthetic, either injectable (PentoBarb) or inhalable (Isofluorane)
  12. Vacutainer brand butterfly cannula; recommended gauge for mice = 23-3/4 [2]
  13. Face mask
  14. Absorbent bench pads (2 per mouse)
  15. Elevated working surface/platform/board
  16. Lab/masking/duct tape

Reagents for isolation

  1. 1x Hank's buffered salt solution (HBSS), without magnesium or calcium, with 0.5mM EGTA [3]; 60-70mL
  2. DMEM-low glucose with 1x Penn-Strep and 15mM HEPES (Digestion medium) [4]; 90mL
    *Make sure the DMEM you use contains calcium*
  3. Isolation medium [5]; 120mL
  4. Collagenase, Type IV [6]; sufficient quantity for 100 units Collagenase Digestive Units (CDU)/mL [7]
  5. Autoclaved water

Materials and Reagents for plating

  1. Type I Collagen, from rat tail; BD #354236 [8]
  2. 0.02N AcAc, sterile filtered, for diluting Type I Collagen
  3. Culture dishes/plates, collagen coated and rinsed with sterile 1x PBS before use [9]
  4. Trypan Blue, 0.4%
  5. Hemacytometer

*A comprehensive materials and reagents list may be downloaded here.

*A spreadsheet listing the media formulations with some simple embedded formulas can be downloaded here.