Hello all,
I want to use Ki67 along with IFN, TNF in relation to T cell inhibitors like PD-1. If I use PBMC's and stim with PMA/iono for 6 hours with golgi inhibitors I am unable to see Ki67 staining (I get great IFN, TNF stains). My questions
1. Is 6 hour too short of a stim for Ki67
2. Should I do longer stim with addition of golgi stuff later
3. Does the fix perm buffer matter? I use the BD fix /perm buffer but is ebio Fox p3 buffer or tonbo buffer better?
Thanks!
Hello Krupa,
here is some (hopefully) helpful advice for your problem.
1. I do think that your incubation time is way too short. I think the average time for T cell division is 12 to 18 hours per division, meaning that's the minimum amount of time you should incubate your cells. As Ki67 doesn't show cell divisions, but rather which cells are cycling I would definitely wait longer. The attached publication shows a time course for Ki67 of human T cells that are aCD3/ aCD28 stimulated (75% after two days) so that would be the minimum amount of time I would wait even with PMA ionomycin stimulation.
Here's the link: http://www.sciencedirect.com/science/article/pii/S0022175910002346
2. If you wait longer you can add GolgiStop or GolgiPlug 3 to 4 hours before you start your staining to prevent cytokine release. I have only worked with mouse cells, but one thing I noticed is that overtly long incubation with GolgiStop or GolgiPlug impedes with cell surface expression of for example CD4 and CD8, therefore a little optimization for the incubation period might be required.
3. I always had great results for intracellular cytokine staining with BD's conventional fix/perm solutions. If you're afraid that this one is not good enough for your Ki67 application I'd recommend the BD Foxp3 buffer, which works great, too. Alternatively, (and only if cost is a concern) try the roughed up home made solution from my old lab
"cells were fixed in 2% paraformaldehyde in PBS with 2% FCS at 37 °C for 10 min, and permeabilized in 90% methanol for 30 min on ice." from
http://www.nature.com/cdd/journal/v23/n5/full/cdd2015140a.html
Works fine, too as you can see in the paper, but maybe it's not as beautiful as using the BD reagents.
4. Lastly as a suggestion. Maybe it's worth labeling your cells with CFSE, which will more accurately monitor, if the cells that produce cytokines also proliferate (assuming that's your question!) There are some issues with CFSE as it stains super bright and spills over other channels especially on the green laser (PE etc.) etc. There are some cool "new" dyes (cell trace violet) that use the violet laser (and make compensation easier), but for that you gotta check your instrument/ flow core, if your flow cytometer is configured for that.
All the best & good luck with your experiments!!!
Michael
Hello Krupa,
here is some (hopefully) helpful advice for your problem.
1. I do think that your incubation time is way too short. I think the average time for T cell division is 12 to 18 hours per division, meaning that's the minimum amount of time you should incubate your cells. As Ki67 doesn't show cell divisions, but rather which cells are cycling I would definitely wait longer. The attached publication shows a time course for Ki67 of human T cells that are aCD3/ aCD28 stimulated (75% after two days) so that would be the minimum amount of time I would wait even with PMA ionomycin stimulation.
Here's the link: http://www.sciencedirect.com/science/article/pii/S0022175910002346
2. If you wait longer you can add GolgiStop or GolgiPlug 3 to 4 hours before you start your staining to prevent cytokine release. I have only worked with mouse cells, but one thing I noticed is that overtly long incubation with GolgiStop or GolgiPlug impedes with cell surface expression of for example CD4 and CD8, therefore a little optimization for the incubation period might be required.
3. I always had great results for intracellular cytokine staining with BD's conventional fix/perm solutions. If you're afraid that this one is not good enough for your Ki67 application I'd recommend the BD Foxp3 buffer, which works great, too. Alternatively, (and only if cost is a concern) try the roughed up home made solution from my old lab
"cells were fixed in 2% paraformaldehyde in PBS with 2% FCS at 37 °C for 10 min, and permeabilized in 90% methanol for 30 min on ice." from
http://www.nature.com/cdd/journal/v23/n5/full/cdd2015140a.html
Works fine, too as you can see in the paper, but maybe it's not as beautiful as using the BD reagents.
4. Lastly as a suggestion. Maybe it's worth labeling your cells with CFSE, which will more accurately monitor, if the cells that produce cytokines also proliferate (assuming that's your question!) There are some issues with CFSE as it stains super bright and spills over other channels especially on the green laser (PE etc.) etc. There are some cool "new" dyes (cell trace violet) that use the violet laser (and make compensation easier), but for that you gotta check your instrument/ flow core, if your flow cytometer is configured for that.
All the best & good luck with your experiments!!!
Michael
That was very helpful. Thanks a lot. I am trying the 18 hr stim with PMA today.
If you're still having issues, you may find this paper helpful: http://www.future-science.com/doi/full/10.4155/bio-2016-0194
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