Hi all,
I'm running seahorse assay on primary hepatocytes to measure mitochondria activity. I always plate each well with same cell number. But they could still be different after 2-3 days of culturing. And it makes no sense for comparison if the cell numbers vary significantly among wells. So I tried two different ways to normalize the cell number:
a. Measuring protein content using BCA assay.
b. Measuring DNA content using Picogreen staining.
However, these two assays gave me different result. I'm wondering:
1. Which assay is better for cell number normalization?
2. Does BCA assay or Picogreen staining also take into account dead cells? If they both do, which one is more accurate in measuring live cells?
Would be great if anyone who had experience could give me some suggestion. Many thanks.
Best,
Surui
Daer Surui!
You look at this protocols, please:
Article Comprehensive measurement of respiratory activity in permeab...
You can also see in thsi review about methods Methods and strategies for normalizing XF metabolic data to cellular parameters
I would suggest you perform BCA assay to measure the total protein content from each well and normalize your seahorse data with it. The alternative way that you can do is fixed your cells after seahorse run with 10% PFA, stain with DAPI and count the cell number with DAPI stain. We performed the cell count with DAPI using the high-throughput confocal microscope.
Hope it help.
Best Regards,
Harry
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