Does any one have a protocol for atrial cardiomyocyte isolation of neonatal rats that they can share?
Our lab is trying to develop and optimize a protocol for the isolation of neonatal rat atrial cardiomyocytes. we already have a protocol for ventricular myocytes but this does not work quite well for atrial cells.
Specifically, we are looking for suggestions regarding
-appropriate culture media,
-plating conditions,
-elimination of fibroblasts and other non-cardiomyocytes
-atrial-specific antibodies for immunostaining
Any help will be appreciated.
Thank you.
Hello
Kindly look at links here
Good Luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770583/
http://www.abcam.com/ps/pdf/protocols/neonatal_rat_cardiomyocyte_harvest.pdf
http://eknygos.lsmuni.lt/springer/374/Part%202/5/2%20Item.pdf
https://www.miltenyibiotec.com/~/media/Files/Navigation/Research/Cardio/gMapplicationnoteneonatalmouseratheart.ashx
Dear Dr. Bolanle Akinwumi
We used Pierce™ Primary Cardiomyocytes Isolation Kit to isolate neonatal cardiomyocytes from 2–3 days old neonatal mouse.
I am not sure if this kit and its protocol would be successful for atrial cardiomyocytes or not however you can have a look, it might be convenient
The protocol that provided with the kit is very easy, herein i will explain it briefly.
You should perform in laminar flow cell culture hood to minimize contamination of isolated cardiomyocytes
1-Heart was isolated and minced in 500 ul of cold Hanks` Balanced Salt Solution (HBSS without Ca2+/MG2+) supplied with the kit.
2-Minced heart tissue was washed twice with cold HBSS.
3-Enzyme mixture was added to heart tissue and incubated in a 37C incubator for 30-35 minutes.
After incubation, enzyme solution was removed and heart tissue washed twice with HBSS.
Next, DMEM (also supplied with the kit ) was added to heart tissue and homogenized by pipetting up and down 30 times.
Finally cells suspension was cultured in six wells plate in DMEM supplemented with 10 % FBS, 100 units/mL penicillin, and 100μ g/mL streptomycin in a 5% CO2 /water-saturated incubator at 37°C.
After 24hrs of seeding neonatal cardiomyocytes, medium replaced with fresh medium containing cardiomyocytes growth supplement diluted 1000 fold.
Cardiomyocytes growth supplement reduces fibroblast contamination and maintain cardiomyocytes at high purity during the cultured period
At day 7 cardiomyocytes purity can reach over 80% .
You would notice that cardiomyocytes start beating after 24hr of seeding
Hope this answer would be useful for you
Best regards
Isehaq
thank you Saleh and Isehaq for the suggestions.
I am still looking for more inputs on atrial myocytes-specific antibodies
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