Hi Kenneth,

I usually use PMA to differentiate HL-60 into macrophages.

If you use DMSO or other polar compounds (like DMF) you will differentiate the cells into neutrophils.

I don't know about DMF but when I am using DMSO to differentiate HL-60 cells, I can see a significant loss of viability from day 6 of differentiation so I usually use the cells on day 5.

How long do you keep your cells in presence of DMF?

Hi Julien,

For now, we keep the cells for 5 or 6 days. We haven't been able to use them for our assays because the viability is so low (though we've tried phenotype and viability staining via FACS according to the attached protocol).

We've optimized getting this guys running, but not their differentiation, so any tips or ideas are very much appreciated.

Hi Kenneth,

I am wondering if your cells are correctly differentiated. Because if you have a HL-60 culture (without adding DMF) starting at 4x10e5 cells/mL I will not be surprised to find 50% loss of cell viability after 5 days if you don't pass them.

Are you cells still growing during the differentiation? (Usually HL-60 stop dividing 2 days after the addition of the DMSO).

Maybe you can check the phenotype of your cells undifferentiated vs differentiated by doing a cytospin + DiffQuick staining ( you can clearly see the change of your nucleus shape in differentiated cells).

You can also check the CD11b expression in your differentiated cells ( I have 70% of positive differentiated cells and 10-15% of positive undifferentiated cells).

Hi Julien,

We do have a planned phenotype check going on, based on the attached protocol. I'm assuming they stop growing, but our confirmations of phenotype were put on hold until we figure out a reason for this viability drop. Though the Diff Quick idea is intriguing and could work well for a quicker time course (though as my group is relatively new, we lack the scope we'd need to view it. It drives us crazy.).

Really, we're just confused as hell on why this protocol says our TB viability should be 90%+ post differentiation, while we're not even remotely close to that value in practice.

Hi Kenneth,

I've attached a copy of a paper in which we use TPA (same as PMA) to differentiate HL-60 into macrophages. In our hands the protocol is simple and reliable. I'm confident that if you follow it exactly, you will solve you problem. If not send me an email and we'll see if I can help you trouble shoot the problem.

Best of luck

Hi Daniel,

That's phenomenal, thanks! I'll look it over later today and talk it over with my boss. The cells we use are just coming up out of froze stocks and are just about ready. I'll definitely give anything a try.

Hi ALL

Without splitting HL-60 cells after 2 days will cause of nutrition depletion. It may the cause of your cell death. I found, HL-60 differentiation with antiapoptotic signals. I am thinking, it may be monocyte or granulocytic differentiation. I did NBT reduction assay. My positive control was 1.25% DMSO for 48 hours. I got dose dependent responses. Can anyone give me an idea about negative control? Is it DPI treated HL-60 cell or 0.1% alcohol trated HL-60 cell? And why we can't use HL-60 alone as negative control?

Hi all,

I'm adding 1.25% DMSO to my undifferentiated HL-60 cells in RPMI-1640 media. I take good care that they don't overgrow and thus die. I often experience that the cells don't differentiate for some unknown reason. Their morphology doens't change and they also lack functions like superoxide generation. Has anyone ever experienced something like this?

Best,

Neele