Your HL-60 cell line may not be what you think it is anymore depending on the source and who started the line and how it has been thawed/cultured initially. Keep in mind that the cells are cryopreserved in 10% DMSO, which also induces them towards the neutrophilic phenotype. If the DMSO is not completely removed when the cryopreserved cells are thawed, they are being induced to differentiate, proliferation slows down and the cells you eventually end up with after weeks of culture, will be unreponsive to DMSO!  Suggest you get new HL-60 cells from a reliable source, follow a protocol which removes the DMSO as much as possible on the first days of culture, and then try again.  I am attaching our protocol for doing this. It works every time! Good luck.  Feel free to share this little know issue with others.

In my experience, HL60 treated with ATRA 1microM are still growing after 5 days, and their cell cycle is similar to that of control cells. At day5, you see an increase of CD11b and CD54.

Personally, I find the HL60 cells a bit tricky. They have to be kept at a density of between 200-500*10e5 cells/mL during and before differentiation. If they grow too dense only once, they won't respond anymore. I've come to prefer NB4 cells which repond much better and are more robust.

Dear Lauren both responses are right however, I think you should do two kind of experiments one compare the differentiation with butyric acid o vitamin D to meke sure your cells differentiate and the other use CD40 as a marker. HL-60 express CD154, but when they differentiate start expressing CD40 losing CD154. I would recommend decrease DMSO final concentration bellow 1 %, 0.1 % is the best. Good luck

I just checked my own protocol again and I used 1.25% DMSO and EMR3/gp91phox as differentiation markers: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2295260/?report=classic That worked very well and I got well-differentiated cells in about 9 days, provided I kept their density below 0.5*10e6 cells/mL.

Thank you all for your responses. I have been treating the cells and re-feeding with treated media on day 3. I was under the impression that post differentiation, the cells would cease proliferation. As mentioned above, it seems I need to maintain the cell density around 5*10e5 throughout treatment. We have a bench flow cytometer. Because this project is in preliminary stages, I haven't been specifically looking at markers. Instead, I run the cells to asses granularity. I see no change. Would the cell morphology be apparent, or do I need to run markers to see if they fully differentiated?

Thank you again!

I guess I should also premise this post, that my goal is to get the cells to differentiate and migrate through 3 micron transwells to fMLP. I also just ordered DMF to try as a differentiation compound.

I have not yet worked with HL60 cells personally, but was told as well that they can be difficult to work with. I used to work with ER-Hoxb8 progenitors, which I found to be a good neutrophil model in terms of their physiology and cellular responses (Wang et al., 2006). If the HL60 cells continued to proliferate post-differentiation, I would be concerned about their maturity - do you know whether this is a specific characteristic of HL-60 cells?

In terms of the granularity - when your cells differentiate you would expect to see a shift for SSC/FSC, but as these parameters can also be affected by cell death and activation, it is always good practice to show maturity through cellular markers. In my lab, we would be using CD11b and CD62L, as well as Wright/Giemsa staining and microscopy to determine the morphology of the cells.

Hope this helps!

Hi Lauren the flow cytometry analysis does not help much in HL-60 cells if they are not synchronized. Recall that the cells may differentiate depending on the factor used. We have seen CD40 expression and loss of Cd154 at day seven with less than 1 % DMSO. If you can stain the cells with Giemsa of Diff quick to check morphology at the light microscope it will be more useful.

good luck

Hi Lauren,

The way we determine differentiation of HL-60 cells upon exposure to DMSO is not through the cell counts but rather through their expression of CD11b by flow cytometry and their ability to release myeloperoxidase upon stimulation with PMA. I've attached a couple of papers that describe in detail how HL-60 cells differentiate upon treatment with DMSO. One of the papers uses PLB-985 cells which are identical to HL-60 cells (see http://en.wikipedia.org/wiki/List_of_contaminated_cell_lines).

Best wishes,

Paige

Hi Lauren, did you solve the problem ? We are encountering similar problem, we cant get HL-60 cells differentiated to neutrophil-like neither with ATRA nor DMSO 

Hi Michal, No I haven't solved the problem yet.  I have tried DMSO, ATRA, DMF, and a combo of ATRA/DMSO.  I have been staining for markers CD11b, CD71, and CD35 on the flow cytometry with no change.  I will run samples today on day 8.  I feel like it could be a technical issue, but I am running out of ideas.  How is your lab treating the cells?  I plan on buying NB-4 cells and see if I have better luck.

Hi Lauren,

Try looking for CD11b expression on earlier days of culture (days 4, 5, 6, and 7). Also, check to see the passage number of your HL-60 cells. If they have been passaged more than 10 times, they will fail to differentiate. I usually replenish my stock of HL-60 cells from ATCC when I encounter problems with differentiation.

Hope this helps,

Paige

Your HL-60 cell line may not be what you think it is anymore depending on the source and who started the line and how it has been thawed/cultured initially. Keep in mind that the cells are cryopreserved in 10% DMSO, which also induces them towards the neutrophilic phenotype. If the DMSO is not completely removed when the cryopreserved cells are thawed, they are being induced to differentiate, proliferation slows down and the cells you eventually end up with after weeks of culture, will be unreponsive to DMSO!  Suggest you get new HL-60 cells from a reliable source, follow a protocol which removes the DMSO as much as possible on the first days of culture, and then try again.  I am attaching our protocol for doing this. It works every time! Good luck.  Feel free to share this little know issue with others.

Hi Lauren. The HL60 cells which we were not able to get differentiated by ATRA or DMSO (experience described in my earlier answer here) come from DSZM (http://www.dsmz.de/). We used PMA induced autochemiluminescence to test the differentiation. We had to obtain HL60 cells originating from ATCC (http://www.atcc.org/). Those work fine for us. See one nice paper which discusses HL60 line variability.

I agree with many of the other comments, in that you should check differentiation by CD11b surface expression within 72 hours.  You need to realize that these cells will never fully differentiate - they are transformed!  Also, remember that ATRA is extremely light-sensitive.  I would also try PMA or 1,25-dihydroxy Vitamin D3 to see if you can differentiate down the other myeloid lineages before declaring that your cells are defective.

Good Luck!

what is source of ATRA and are you dissolving in etoh or dmso to prepare your stocks for further dilution in growth media?

Hello,

Just wondering when I stimulate the HL60 cells with DMSO and use them around D7, do you advise to change the medium just like how we normally pass down the cells? Or should I keep them in the same medium until I use them?

And whenever I change medium is it necessary to add DMSO? 

Because I found that most of my cells were dead if I don't change the medium over time

Can somebody advise me on this?

Thank you

Just keep the same medium with DMSO until you use them. You should wash off the DMSO by repeated centrifugation in your assay buffer before using them.