Hello,
We're going to perform a Seahorse assay using XFp 8-well system. The cell density will be 20,000 cells/well. We don't have the Cytation system to perform automated cell counts. As well as, the cell number is too low to perform a BCA assay (Pierce from Thermo Fisher). What other methods should we try to normalize the data? We will appreciate any recommendations.
Thanks,
Samina.
Could you do some low magnification imaging of the cells, specifically fix the cells and stain with DAPI and count nuclei in your images to determine an estimate of cell number?
Perhaps also a luminescent ATP viability assay? May have to add the reagents to your 8-well plate and then load into a 96 well plate to run in a microplate reader.
Hi Aaron Dhanda,
Thank you so much for your reply! Actually, we're also thinking of fixing and DAPI staining. But I have a couple of concerns about fixing and imaging. We would appreciate it if you have any suggestions regarding these concerns.
1. What type of fixative are you suggesting? 4% PFA or ice-cold methanol for 15 min?
2. About imaging, say I am going to use 10x objective, I can't get the images of the whole well. Do you have any suggestions on how I can reciprocate this count for the whole well?
I will also look forward to the ATP viability assay it as well.
Best,
Samina.
Dear Samina Momtaz
4% PFA incubation for 10-15minutes is suitable for fixing most cells and for DAPI staining this would also work.
you can acquire images from 5 different regions (top, bottom, left , right and centre) for each well and this will be a decent representation of the overall cell density.
you can try the cell profiler tool (https://cellprofiler.org/ ) for automated quantification of DAPI signal in case you have too many images and manual counting gets challenging
All the best
Hi Samina,
1. I would guess you should be able to fix your cells with 4% PFA (prepared in PBS -/-) for 15 min at room temp. I like to pre-warm my PFA to 37c (or whichever temp your cells are growing at).
2. Yes, given the size of your well (8 well), you wont be able to image the entire well in one field of view. I would say decide on a number of images per well that you could use for statistical analysis (at least 10 images/well is a good starting points, it's a bit arbitrary in how many images you would exactly need). If possible also try to take images that are representative of the well density. Once you have your images, you will want to analyze them with image J or Fiji. Search on google something along the lines of 'counting nuclei using imageJ' and you should be able to find a guide on counting all the nuclei in your images. There are several ways to do nuclei counting so pick one you can easily do. Then I suppose you can just normalize your nuclei counts of your experimental/treatment wells to the nuclei counts in your control well.
Hi Aaron Dhanda ,
Thank you so much for your suggestions. I will follow your protocol.
Best,
Samina.
Hi,
You can also use crystal violet to dye your cells. Afterward, you add acetic acid to dissolve the crystals and you measure the DO.
The DO will positively correlate with the amount of cells.
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