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Heart Tissue Dissociation

Tianfang (Frank) Yang; Email: tyang5@tulane.edu
Hua Li; Email: hli28@tulane.edu
Ph.D. Student
From Yiping Chen’s lab
Email: ychen@tulane.edu
Tulane University
Department of Cell and Molecular Biology 6400 Freret Street
2000 Percival Stern Hall
New Orleans
LA 70118

Heart Tissue Dissociation

  1. Transfer tissue into 1.5mL or 2mL microcentrifuge tube.
  2. Wash tissue with PBS for 3 times.
  3. Mince tissue into small pieces until no more than 1mm.
  4. Add 1mL 2% collagenase I, II, IV to the tissue.
  5. Incubate tissue at 37°C for 10 minutes, then pipette gently to disperse the large pieces of your tissue.
  6. Repeat 5th step for 3 times, then check it. If there are still some large pieces, centrifuge it for 5 at 4°C.
  7. Incubate tissue at 37°C for 10 minutes. Then check it until all of the tissue are dissociated into single cells.
  8. Stop digestion by using 1mL 10% culture medium. (I prefer to add 1mL fetal bovine serum)
  9. Use 40um cell strainer to filter your cells and transfer cells into a 15mL Centrifuge Tube. Spin cells for 5min at 250x g at 4°C.
  10. Use 400ul 10% culture medium to suspend cell pellet.

Material
1. 2% collagenase I, II, IV: add 0.04g collagenase I (17018029, invitrogen), collagenase II (17101015, invitrogen) and collagenase IV (17104019, invitrogen) to 20mL HBSS (14175095 thermo)
2. StemPro® Accutase(A1110501, Fisher Scientific)