I already did some activation experiment to observe expression of CD40, CD86, CD80 and MHC with macrophage 264.7 and dendritic cells (DC2.4). I treat these cells with positive control LPS and therapeutic nanoparticles or bare nanoparticles. So, once I collected the cells, washed them and fixed with 4% paraformaldehyde (pH=6.9) or 5% Formalin in PBS. But I observed that I was losing cells with washing, fixing and post-fixing washing steps. My events/second in the flow cytometry were very low. I was thinking what if I don't fix the cells and I was wondering what was the purpose of fixing?? If I don't fix the cells, should that affect the results negatively? Thanks
Hello Sudip Kumar Dam,
If you don't want to fix the cells, then you should try to analyze the cells on the flow cytometer as soon as possible, within an hour's time. If the cells are not analyzed in the flow cytometer immediately after antibody staining, then you may have to fix the stained cells with 1-4% PFA, 20 mins at 4°C. For extended storage as well as for greater flexibility in planning the time on the flow cytometer, you will have to resuspend the cells in 1-4% paraformaldehyde to prevent deterioration.
For surface antigens, you should stain the cells and then fix, if you are not going to carry out the analysis immediately. However, if your target is intracellular, you’ll have to fix/permeabilize the cells prior to staining anyway allowing the antibody to reach its target. The controls will require fixation using the same procedure.
Fixation is good for cells labeled by fluorochrome-conjugated antibodies to membrane antigens. It will stabilize the light scatter and labeling for up to a week in most instances, allowing you to be more flexible in scheduling your flow cytometer time. Also, it inactivates most biohazardous agents.
Best.
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