Hello, I performed a titration of FCCP in HEK cells and two other melanoma cells lines in order to do a mito stress test with the Seahorse technology. While I can clearly see an increase in OCR with 0.5µM FCCP for the two melanoma cell lines, I can't see any increase in HEK cells (neither with FCCP 0.125 and 0.25µM) and a decrease with FCCP (1 and 2µM)... Has anyone encountered this situation with HEK cells? and what can be done about it?
Thank you!
Hi Sara,
No, I never obtained a good response to FCCP with the HEK cells. And I didn't pursue the experiments with this cell line. Strangely, I observed that the cells changed morphology after adding the assay medium - even if I supplemented with the correct amount of substrates and adjusted the pH to 7.4.
All the others cell lines I was using - melanoma cell lines (A-375 and Mel-Juso).
I experienced a similar problem with HEK cells where the response to FCCP did not scale with the concentration curve. Did you check the wells and port injections after running the assay to make sure all injections were successful and complete? I realized the port injections were not always successful - presumably due to air bubbles in ports or issues with the actual machine during injections. I never use a multi-channel to fill ports on the 96 well plate to avoid air bubbles forming and I had success with 0.5 - 1.0uM FCCP with HEK293T cells. Since I usually have 6 minimum data points per cell line/condition tested and if I see a port did not inject I just remove the data point.
In my case, when I saw the cell did not give good results ( good curve), I usually change cell number ( so I optimize the cell number I will seed for each cell line).
Your basal level is low. I would add more cells or do a titration. You should aim to have a basal level OCR around ~100-150. Once you have your cell number optimized, do another titration for FCCP. For my rat heart cells, the optimal FCCP concentration is 7 uM (just to give you a reference)
Hope this helps. Good luck and keep us posted.
In my hand, 0.5 and 1 uM FCCP worked in 96-well format assay for HEK293 cells. Usually 10K cells is suitable in 96-well format (Make sure your basal OCR falls within the range as mentioned in Seahorse) . Use the MitoStres kit as well as pH adjusted medium from Agilent. Pay attention your pipetting too. Additionally, you need at least 16-replciates per group. You can skip a couple of data points in case if you get negative value/outliner. Last but not least, normalization is essential. Need to do cell counting for 96-well plate for data normalization. Hope this helps.
My experience is that hek cells will be suspension (hardly attached) when I was normalizing the cell number under cell cytometry.
For the cells that are not so adherent, it is a good idea to coat the plates (with poly L-serine?) or the cells will be suspension after mixing lol..
I use HEK293 and get a good FCCP response. A couple of pointes for HEK293 cells respiration assay:
1) As Yuanyuan mentioned, HEK293 are loosely attached to the plate, so it's always a good idea to coat and then seed the cells.
2) For 24 wellplate, I seed 70,000 cells. Optimize your cell number for 96 well plate to get maximum response.
3) For me, HEK293 cells give a good response at 1 uM FCCP concentration.
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