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
- Transfer tissue into 1.5mL or 2mL microcentrifuge tube.
- Wash tissue with PBS for 3 times.
- Mince tissue into small pieces until no more than 1mm.
- Add 1mL 2% collagenase I, II, IV to the tissue.
- Incubate tissue at 37°C for 10 minutes, then pipette gently to disperse the large pieces of your tissue.
- Repeat 5th step for 3 times, then check it. If there are still some large pieces, centrifuge it for 5 at 4°C.
- Incubate tissue at 37°C for 10 minutes. Then check it until all of the tissue are dissociated into single cells.
- Stop digestion by using 1mL 10% culture medium. (I prefer to add 1mL fetal bovine serum)
- 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.
- 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)
