I have not tried it without coating solutions. I had good success by placing 4x10^5 cells in 12 well plate with Supplemented Claycomb Medium, in the absence of norepinephrine and penicillin/streptomycin. On the day of the transfection, for EACH well of a 12-well plate, dilute 2 ug of DNA in 100 ul of Opti-Mem (no serum or antibiotics). For each well, dilute 5 ul of Lipofectamine 2000 (LF2000) in 100 ul Opti-MEM (no serum or antibiotics). Gently mix the complexes. Incubate for no more than 5 minutes at room temperature. Combine the diluted DNA with the diluted LF2000. Mix gently and incubate for 20 minutes at room temperature. Add 0.8 ml of norepinephrine-free and penicillin/streptomycin-free Claycomb Medium (with serum) to each 0.2 ml of DNA/LF2000 mixture. This constitutes the Transfection Medium. Transfer 1 ml of Transfection Medium to each appropriately-labeled well. Incubate the cells at 37°C in a humidified CO2 incubator for 18 hours, at which time add 1 ml of norepinephrine-free and penicillin/streptomycin-free Claycomb Medium (with serum) per well. Replace the Transfection Medium with norepinephrine-free Supplemented Claycomb Medium 24 hours after the start of transfection.

Dear Chad,

Thanks very much! I really appreciate your detailed protocol which I will surely try. I reckon that your transfection efficiency is higher than ours (around 15 %). In our case, we add double LPF in less time of incubation (3-6h) because our transfection medium is just OptiMEM.

Dear Chad,

I'm trying to transfect HL-1using your protocol. Concerning protocol I have a doubt. 24 h after transfection do you replace medium with Claycomb complete with or without norepinephrine? If it's without, why is it?

Concerning results: I've just checked the cells 18h ours after the transfection and looked like there is no much transfected cells...Maybe it's too early to see full expression but anyway...Which is your transfection efficiency using this protocol?

Many thanks again!

Sorry Selma, I just noticed your reply. I had a transfection efficiency of ~50% based on the RFP fluorescence. Everything is without NE. This was based off of previous observations in the literature. Gene knockdown in my case for Orai was seen at 48h and cells were tested 72 h following transfection.

Thanks a lot, Chad. The truth is that my real truble was the fluorescent tag plasmid I was trying to transfect (with DsRed) that didn't work properly. Morover, HL-1 cells have red autofluorescence and that was increasing troubles. I made tests using GFP following the new LPF2000 manufacturer's instructions. The effciency was surprisingly high! Following their new protocols saves lot of time and reagents. Check it: http://www.lifetechnologies.com/content/dam/LifeTech/migration/files/cell-culture/pdfs.par.6683.file.dat/lipofectamine%202000%20reagent%20protocol%20v2.0.pdf

P.S.--I think we are in the same league now, struggling with mouse ventricular myocyte patch clamp -saw your question ;)

Dear Chad and Selma

I'm trying to stablish HL-1 culture in the lab. I'm working currently. I'm really excited, today I had my first beating cells.

my next step is transfect them. I culture them into 2ml nunco dishes (coated). To transfect them, do you wait until they beat? or before they start?

Thank you very much!

pS: Very interesting topic :)

Glad to hear it all worked out Selma.

Dear Beatriz,

Congrats for your HL-1 cell culture, wish you all the best!

What do you mean by waiting until they beat? I usually defrost a batch of cells (as young as possible) and leave them grow until they reach confluence. After the first passage they are ready to be transfected. That's my own experience. They never stop beating, I guess. Hope that helps!

Dear Selma,

I have just started working on HL-1 cells and unlike what you mentioned above, my cells in the culture do not beat, i.e, contract. Is this some problem with the cell line I am using? I also would like some further help on this matter, if you have time.

Thanks.

Prabodh - Are you using Norepinephrine prepared in 30 mM ascorbic acid? The final concentration of NE in your medium should be 0.1 mM. What is your confluence when you split and plate? Did you receive the cells from William Claycomb or another investigator?

Chad- I am using Norepinephrine prepared in 30 mM ascorbic acid and the final concentration is 0.1mM. The problem I think is not of the confluence because I make sure that the flask is really confluent as adviced by the claycomb lab. The cells however were received from another lab who primarily had received it form Dr. Claycomb.

Hi Pradobh;

I experienced that when the passage is too high they have problems to beat. also Dr. Claycomb recommends to use specific lot of FBS. Last time I bought it from Sigma N° F2442 and it worked.

If they still don´t beat, I would recommend you to get newer cells.

Good luck.

Thanks Beatriz, you got my name wrong though :). What lysis buffer do you use for protein preparation for western blotting using HL-1 cells?

Beatriz brings up a good point with the FBS. I would also get the cells from Claycomb directly. As for protein preparation we use Cell Extraction Buffer from Invitrogen (FNN0011).

Thanks Chad. I just wanted to know if the cells retain their cardiomyocyte properties of action potential and calcium transients sans the contracting nature.

Make sure they are mycoplasma free, we've had mycoplasma HL-1  cells which always showed 40% GFP

Thanks a lot Selma

Hi! Im doing a master degree in CNB and the person who is instructing me in the lab did try to grow HL1 cells without coating. She got good results but with a little difference in appearance of the cells for a couple days. So the cells did grow consistently normal for a couple days but after that the cells started to grow really bad. So indeed the coating is necessary for some reason.