Hi can you help me, which blocking buffer recipe can one use for isotype control on platelets, can you share it with me? I'm doing flow cytometry on blood platelets and I'm using conjugated antibody which is CD41a-APC and CD62P-FITC. I'm adding 20ul each for staining. For the isotype control which is my negative control I'm using an unconjugated primary antibody which is mouse IgG1 and conjugated secondary antibody which is Alexa fluoro 660 goat anti-mouse IgG. I'm adding the primary antibody at the same concentration as the positive controls antibodies which is 20ul . My second question is, should I add the same concentration of secondary antibody too as the positive control antibodies and isotype control, or it should be different? If its different what concentration can you recommend for staining?
thank you
Hi Millicent,
First of all, if the markers are expressed on cell surface, you don't need a blocking buffer but if the marker is intracellular you can incubate your cells for 20 min on ice with 100% FBS. Yes the concentration of the IgG control should be the same with the positive one. 20 µl is a bit much. 1 µl of antibody for 5 million cells should be enough for 15-30 min incubation on ice..
Best wishes
In general, if your experimental Ab (what I believe you call "the positive control antibody") is a fluorophore-conjugated Ab i.e. no secondary Ab is used/needed to detect it, your isotype control Ab should also be conjugated to the same fluorophore as the experimental Ab.
I agree with Vladimir, you really need to get some isotype matched antibody that is labelled with the same fluorophore as the antibody of interest.
I didn't think that you mentioned your stain buffer, is it 3-5% FBS in HBSS or PBS? do you use Fc block (anti-cd16/32)? Perhaps more detail about your protocol would help people understand what you are trying to do.
also, please keep in mind that you can't compare volumes of antibodies without knowing the concentration of the solution you add, check on the insert or with the manufacturer the concentration of IgG to be sure you really add a corresponding amount of your isotype control antibody. And, as Vladimir says, you need to buy a directly conjugated isotype antibody as control for directly labelled antibodies. Also, in that case, you need to match the F/P ratio of the isotype and specific antibody, as especially FITC-labelled antibodies may be labelled with different numbers of FITC molecules per antibody, so to assure a corresponding fluorescence in the negative sample, this should also be matched.
I agree with Sofia that the number of fluorophores per antibody is crucial. Companies will not usually indicate the stoichiometry of the antibody, so you usually have to contact them to figure this out as well as titrate the control and experimental antibodies. Also, 0.1 - 1.0 % bovine serum albumin in your antibody dilution buffer is usually a good idea to reduce nonspecific interactions.
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