Improve Research Reproducibility A Bio-protocol resource
bpdetail_icon_lock
Preprint

Isolation of single cells from cryoinjured hearts

JB Jeroen Bakkers

Last updated date: Dec 5, 2020 Views: 897 Forks: 0

original An abbreviated version of this protocol was published in eLife in Dec, 2019
Single-cell analysis uncovers that metabolic reprogramming by ErbB2 signaling is essential for cardiomyocyte proliferation in the regenerating heart
download pdf Download PDF
ask Ask a question
cite How to cite
nfavorite Favorite

* Keep everything at room temperature until FACS sorting, afterwards cells can be put on ice.


Protocol:

  1. Prepare HBSS + Heparin mix (Ca2+/Mg2+ free, Sigma #H9394).

  2. Isolate ventricles and dissect hearts in HBSS + Heparin mix. Hearts should still beat, try to remove all excess tissue such as pericardial sac, fat and blood. 

  3. Collect hearts in 2ml eppendorf tube (ideally 5 hearts, but ok with 7) and rinse in 1x dPBS

  4. Digest hearts with 1ml collagenase 1mg/ml, resuspended in HBSS (Ca2+/Mg2+ free), on shaker at 32 degrees, 700 rpm for 30-40 minutes. Pipette hearts up and down every 10 minutes to facilitate breakdown of the heart. 

  5. Spin 2000 rpm for 5 minutes at room temperature.

  6. Remove supernatant (make sure you don't disturb the pellet)

  7. Add 1ml TrypLE express, on shaker 32 degrees, 700 rpm, 15-30 minutes. Pipette every 5 minutes up and down until single cells are obtained.

  8. Prepare 4x volume of complete media (1ml/tube). Complete media = 20% FBS in HBSS (Ca2+/Mg2+ free).

  9. Inactivate enzyme by adding 1 ml of complete media and strain mixture through 70 um cell strainer into a 15ml tube. Add another 1ml complete media to eppendorf tube to wash and put through strainer, repeat until 3ml complete media is added. 

  10. Spin 15 ml tube for 5 minutes in a rotating angle centrifuge at 2000 rpm at room temperature. 

  11. Discard supernatant, wash with 1ml complete media and spin for 5 minutes 2000 rpm.

  12. Resuspend pellet in 500ul complete media

  13. Strain cells through 40um strainer

  14. Leave at room temperature in the dark.

  15. Just before sorting, add viability dye. This can be DAPI 20ug/ml, 1:200 dilution; or PI 1mg/ml (Sigma P4864), 1:500 dilution. 

  16. Sort cells. 

Copyright: Content may be subjected to copyright.
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Bakkers, J(2020). Isolation of single cells from cryoinjured hearts. Bio-protocol Preprint. bio-protocol.org/prep682.
  2. Honkoop, H., de Bakker, D. E., Aharonov, A., Kruse, F., Shakked, A., Nguyen, P. D., de Heus, C., Garric, L., Muraro, M. J., Shoffner, A., Tessadori, F., Peterson, J. C., Noort, W., Bertozzi, A., Weidinger, G., Posthuma, G., Grün, D., van der Laarse, W. J., Klumperman, J., Jaspers, R. T., Poss, K. D., van Oudenaarden, A., Tzahor, E. and Bakkers, J.(2019). Single-cell analysis uncovers that metabolic reprogramming by ErbB2 signaling is essential for cardiomyocyte proliferation in the regenerating heart. eLife. DOI: 10.7554/eLife.50163

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

0/150

tip Tips for asking effective questions

+ Description

Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.

post Post a Question
0 Q&A
This protocol preprint was submitted via the "Request a Protocol" track.