Hi Seuk-Un,

First of all, I just want to ask your Raw cells morphology is really nicely round and you can detach easily just by pipetting. You need good Raw cells like this. And, maybe Raw cells seem like float during differentiation and fusion each other for making osteoclasts. I usually use 1000-2000 cells/well in 96-well plate (you can caculate optimal cell number for 12-well plate by area). Cell number is really critical when you make osteoclasts.

You already told, your cytokines were Ok with primary bone marrow cells, so I don't doubt that your cytokines is kind of toxic to cells.

And final thing is, actually Raw cells don't be needed M-CSF for osteoclastogenesis. Those cells are already manipulated in M-CSF receptor that is activated always, so you don't need to add M-CSF. Please only add RANKL, you can test RANKL dose-dependent or cell number different.

Let me know if you have more questions.

Thanks,

J

Jungeun Yu

I'm really appreciate to your reply.

Testing the concentration of RANKL is the best method for now.

can I ask you one more question about the cell concentration?

I know that a 12-well plate has an area of 4cm^2 per well and a 96-well plate has an area of 0.32cm^2 per well.

The cell concentration that I tested is much higher than the 1x10 ^ 4 in 96well plate. I think that the distance between the cells should be short enough that the fusion will work well, so I experiment with a higher concentration. In the primary cell experiment, this concentration was good, so the raw cells also progressed to the same concentration.

Could this high cell concentration be a big problem?

I will attach the picture to show the shape of the cells. There are few of strange shaped cells, but most of the cells are round. what do you think?

Hi,

RAW cells grow faster than primary macrophages/monocytes, and thinner cell density is necessary at the plating of the cells. Or RAW cells get overgrowth, which results inhibition of the multinucleation.

In case your uploaded image of RAW cells is the image of just after plating of the cells, it may be too much number of cells. Try 1/10 cell density.

Hiroyuki Kanzaki

Indeed, the raw cell grow really fast that I have to subculture every 2 days.

So, lower the seeding concentration is make sense.

Thank you for your opinion!

Attached image is not seeded, but a raw cells that is culturing. (actually, those cells was melted yesterday. I will subculture tmr.)

Thanks again!

Hi Seuk-Un,

Yes, definitely you need much less cells for osteoclastogenesis with Raw cells. I used 1x10^3 or 2x10^3 cells/well of 96-well plate, not 10^4 cells, I know, much much less cells than yours. You can test various different cell number.

Cells look not the best. I cultured and split Raw cells by pipetting, because I want to do gently, and get only round cells from stretched cells. I usually didn't do trypsinization.

One more thing, I had experience that some Raw cells were differentiated well, some cells were not (I had several different batches). If you have different batches or passages, you can test.

Good luck!!

J

Jungeun Yu

I use a scraper instead of trypsin to subculture. I heard trypsinazation can activate the raw cells, so I never use it.

I will try several concentration of both cells and RANKL.

Thank you for your comment!