Dear all,
I would like to know if any of you is having issues with the Naive CD4+ T Cell Isolation Kit II, human from Miltenyi Biotec or with the CD3/CD28 activation beads for naíve CD4+T cells from Gibco. For a long time, my naíve CD4+ T cells don't get activated when cultured with Dynabeads and x-vivo 15 media. I also noticed that the yield of naíve CD4+T cells I get is lower than before.
I already tried several things (like increasing the beads ratio) but nothing seems to work. It would be a greta help if anyone is experiencing the same problem.
Hi Laura,
Are your CD3/CD28 activation beads new? If not, I suggest buying new one for your experiment. In my experience, the activity of activation beads decreases over time (Maybe the vortex process somehow damage the Ab-bead binding). You may need to add two fold of the old beads to reach the efficiency of the new ones. So I always titrate bead activation activity for a certain interval.
they are not expired... I even tried 1:2. There was cumping but the media color (which has phenol red) did not change color.
Bead activity decreses even it is not expired. How do you determine the cells are activated or not? What’s your readout? Cell proliferation? Cytokine production?
If I'm lazy just count the cells (with trypan blue) and look a them in the microscope. There should be clumping and the color of the media should turn from red to yellow after 120h incubation.
I eventually did flow checking CD25 and a viability dye.
I think you could set some positive controls, such stimulating cells with aCD3 and aCD28 Ab, in this case. The positive control can help you check if the beads work or not. As I mentioned above, the activity of the bead decreases over time.
I highly recommend you checking cell proliferation as readout. Try labeling cells with CFSE or other proliferation dyes.
Is there anything wrong with the buffers? Please use new buffers and a positive control.
I prepare fresh buffer every time for the isolation.
Actually for my experiments cells with beads are the positive control since I treat cells with a toxin and inhibitors.
I could try to use CD3/28 Ab to stimulate the cells and use that as positive control.
Yes, please use CD28/CD3 abs to stimulate your cells. You can also run an exp. with x-vivo vs RPMI 10% FBS. We had some similar issue with a bottle of x-vivo in the past.
If you have low numbers of naive CD4+ cells then ensure you culture them in a u-bottom 96 well plate at a concentration of 1 million/ml and 200ul per well, plus 100IU IL-2 and recommended CD3/CD28 activitors.
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