Does anyone have such experience? How accurate can it be? Anything to be noticed during the procedure? Any good alternatives?
I assume that you are refering to counting by Neubauer like chambers. I think critical step for counting fibroblast like cells (by any means) is efficient trypsinization. They tend to form aggregates making it hard to have reproducible counts. If you are working with confluent cultures, it becomes more problematic. May be giving a trypsin wash before trypsinization, longer trypsinization and not shaking the plate in between could help. We do it always and I dont think you need to search for alternatives. Good Luck.
You can use a "graticule" to count the cells. This is an microscope eyepiece that has an etched chamber of distinct size so that you can count the cells. My advice to you when counting cells is to get another colleague to do this as they will not know the cells or the experiment you have set up i.e. to reduce introducing bias to your results
Dear Yanshen,
For adhering fibroblasts, I first perform a stringent trypsinization. During this process, the cells detach from the bottom of the culture flask and become round-shaped. As mentioned before, fibroblasts tend to form aggregates, thus it´s crucial to also pipet up and down a couple of times while taking them up after trypsinization, to obtain a single cell suspension. From there, it´s standard counting. I used to count my cells 2 to 3 times, and am still doing it if it´s crucial to have an exact cell number, and always get close-range results. So, in my hands, it´s not difficult if the trypsinization goes well!
Hope that helps! Best, Julia
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