PBMC show different morphology after cona stimulation
one (patient 1-1,1.1,1.2,1.3) showed proliferation and clear cell boundary.the first 4 pics.
:pbmc 96 eh c5, pbmc hk 2, pbmc hk, pbmc.
but the other (patient 2-2,2.1,2.2,2.3) showed no clear proliferation cluster, and scatter almost whole well without leaving any space. and cell show no clear boundary:
what's the reason?
Hi Ming,
Individuals from any population have variable responses upon stimuli. Especially if you're working with patients. What kind of disease are you working on?
Hi Ming! I agree with Shubhranshu. Do you count your PBMCs before you set up your experiment? Always start with an equal number of PBMCs in each well/tube. Very low lymphocytes count in the second flow cytometry pic may be due to less number of PBMCs from the start in the patient 2. And try to break the PBMC clusters into single cell before adding them into the well/tube otherwise, you will lose the cells in the singlets gate (FSCA Vs FSCH). For the morphology aspect, sometimes the reagents that we use for the PBMCs isolation can be toxic to them (eg., Lymphoprep).
We keep our isolated PBMCs in isopropanol (-80 degree) overnight and next day we transfer them to liquid nitrogen. After thawing, we get 50-60% viable cells.
Hi Ming,
We count them under inverted microscope using Trypan blue (0.4%, Sigma).
Ming Lu Sorry for reactivating the post after such a long time. But I was wondering what you meant by good patient and bad patient.. Actually, Today, I came up with similar phenomenon as your bad patient.. I used LPS and ConA to stimulate the PBMCs from pig. with the LPS the FSC-SSC profile was similar to un stimulated cells (non-treated). However, the cells treated with ConA (5ug/ml) has lower FSC and Higher SSC as seen in your bad patient. for The FSC-SSC graph, I had only one population on the left for conA group. compared to two populations (one of which for sure is debris) in non-treated or LPS group (as of your good patient). the cells were treated at the same time and had similar profile. so this shift in FSC-SSC profile is something because of con-A (for sure). I wonder if you did sort that out.
Do you think this shift might be because of death of the cells. I forgot to use viability dye?
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