I've been trying to differentiate RAW 264 cells into osteoclast and count them in 96 well plate. But I'm not sure about how other researchers count no less than 100 osteoclasts in a well under the light microscope. Do you count such many osteoclasts VISUALLY, or do you count by using some software?
Although I tried to find the way to count osteoclasts, but I couldn't get useful information. (They just describe like that "count TRAP-stained multi nuclei cells using light microscope.") Please help me.
Hello there,
Number of osteoclasts depends on how differentiated the osteoclasts are since they tend to merge and become giant multi-nuclear cells. So I probably wouldn't worry too much about getting hundred plus counts. Acid Phosphatase is characteristic of mature osteoclasts and is stained by Trap b staining. You shall see easily distinguishable big pink/red/maroon colored multinuclear cells under regular light microscope (differentiated osteoclasts). Then, you can count them manually (which is more accurate in my opinion although tedious) or you can image it using systems like Cytation5 and count it there.
Hope it helps. Good luck.
Hi Mizuki,
I totally agree with Sudipta when it comes to counting RAW264 cells. As far as I know these cells proliferate fast compared to others (manual counting will take a lot of time).
It is a pre-OC cell line, not sure if you will see matured or multinucleated osteoclast differentiation as you observe it in ex-vivo experiments.
Amit Chougule, Sudipta Baroi
Hello my teachers,
Thank you for your kind advice. I think RAW264 cells are too much to be imaged by using software, so I'll count them manually. If you know some ways to count cells easily and objectively, please let me know.
Thank you so much!
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