I am working on effect of HIV-1 on phosphorylation of cytoplasmic proteins to be analyzed by flow cytometry. For staining purpose it is recommended to permeabilize the PBMCs with Perm III buffer (BD) on ice. But instead of incubation on ice, can I incubate cells at -20° C with Perm III buffer?
Imagine a cell, what it is, what are the major components of cell. It is a little bag of fine structures in water environment. If you throw it in a freezer, you destroy it.
Incubating cells at -20C will cause total disruption and you won't have any cells to run through the flow machine, just a lot of debris.
As per my experience, the experiment can be performed on ice bath. However, the permeabilization should be done under gentle shaking with recommended agents or Triton X.
Hey Ashwini,
I would rather suggest you to use 4 degree instead of -20 degree. I have an experience. you should try it once with healthy control.
Thank you all...will follow your suggestions during my next experiments...
I agree that using a -20° temperature can be very dangerous for cell integrity
I think -20o degree is definitely dangerous for cell, I advice you put them at 4o degree and shake the cells every a few minutes.
I guess that using minus twenty degree is not convenient since congelation followed by decongelation will break cell membrane and probably kill at least part of them.
The best way is to incubate on ice or refrigerate (4oC). -20oC is not allowed as it may damage the cells. The time is also very critical and it is dependent on the application.
Hi Ashwini,
Using -20oC with live cells would normally be a bad idea. However, Perm Buffer III from BD is methanol if I am not mistaken, and I assume that you will fixate your cells with formaldehyde before you start permeabilizing them, and in our hands it is perfectly fine to permeabilize PBMCs with 90% methanol at -20oC. We based that approach on the protocol here: http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.10072/pdf that was developed by the Nolan lab, who has used phosphoprotein profiling very successfully. On page 63 of the above-mentioned journal (Cytometry A. 2003 Oct;55(2):61-70; see link) under "Phospho-epitope Staining for Flow Cytometry, Optimized protocol" you can read the following:
"They were then permeabilized by resuspending with vigorous vortexing in 500 l ice-cold MeOH per 106 cells (this is an approximate number, and more or less methanol can be used as long as evaporation is not significant) and incubated at 4 °C for at least 10 min. At this point, cells can be stored at -20°C for several weeks with minor loss in signal strength."
The protocol, which we developed is an adaptation from the protocols as developed by the Nolan and Jacobberger labs because we stain most surface markers after fixation but before permeabilization, can be found here: http://www.ncbi.nlm.nih.gov/pubmed/20956346 (see paper), and the same protocol was used to study induction of phosphoproteins after IFN-beta injection of patients, here: http://www.ncbi.nlm.nih.gov/pubmed/22106296 (see paper). Other groups have also used our protocol successfully, as you can see here: http://www.ncbi.nlm.nih.gov/pubmed/23872679 (see paper).
Basically, we fixate our PBMCs or whole blood with 2% or 4% formaldehyde (end concentration), respectively. It is very important to use methanol-free formaldehyde (i.e. 10% Formaldehyde, EM-grade, methanol free, Polysciences, Worington, PA) if you need to stain certain surface markers before your permeabilization step to enable identification of individual leukocyte subsets, because methanol can potentially destroy surface epitopes (you will have to verify that yourself: BD has a website with anti-CD antibodies that can be successfully used after pemeabilization with methanol or Perm Buffer III). After fixation and staining of surface markers, the human cells are permeabilized by adding to each tube 1 ml of ice-cold 90% methanol in 1xD-PBS while vortexing at high speed, and are typically incubated O/N at -20oC. The minimum incubation we did at -20oC was 1 hour; and the maximum incubation we did at -20oC was 3-4 days without loss of signal (please note that this also depends on which fluorochromes you are using, thus you will have to verify this for your application). Nevertheless, we never tried permeabilization with Perm Buffer III from BD at -20oC; besides the methanol (not sure which concentration) I do not know what is in there, thus you would have to test if it is OK or even better for your application to incubate at 4oC or at -20oC. If you have any questions about my protocol, you are very welcome to contact me at [email protected]. Best of luck with your experiments.
Thank you Anette...this is really a good information...but is there any specific grade of methanol to be used for permeabilization?
We always used a high-grade methanol (99.8%, Mallinckrodt Backer Inc, cat# 9070-07).
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