Hi Sergio,

I will try to help with some of your questions since you have so many. You can mix as many antibodies as you can, since your fluorocromes don't overlap, you need to be aware of that. It doesn't really meter the companies that you buy the AB from, but is good to know the concentration recommended and I suggest ti titrate the AB before you use it so you can use an ideal amount. Some times the companies extrapolate the concentration just to play in the safe side, so you may be able to use less. Buffers for flow can be: PBS+BSA or PBS+FBS and a lot of people add EDTA to those, since some cells form clusters and EDTA will help with that. Clusters may block the cell acquisition during your flow analysis. Personally, i think that adding EDTA may help.

I know that i haven't answer all your questions but i hope that I was able to help some.

Erika

Hi Sergio,

I perform flow cytometry in 96 well plates, and I use 100,000 cells to 200,000 cells.

I resuspend them in 100 ul of Buffer. Regarding the concentration of antibodies, I agree with Erika, you must titrate them to know the appropriate amount to be used.

Buffers for surface/extracellular staining: Buffer 1:  0.5% of BSA+ 3mM EDTA + 1 litre PBS(w\o Ca & Mg)

Buffers for intracellular staining: Buffer1, & Buffer 2

Buffer 2: 0.5% of BSA+ 3mM EDTA + 1 litre PBS (w\o Ca & Mg)+ 0.1% Triton X 100

I have attached two protocols. These work for my cell types.

Hope it should work for you.

All the best

Santoshi

Very nice! thank you very much. I only have one doubt more:

I have everything more or less clear, but I realized I do not have the rotors for 96-well plate. It does not matter because I do not have too many tubes, however my principal doubt are the number of cells.

In protocols that I read they say: Adjust cell suspension to 10^6 cell/ml and take alicuots of 50 ul to mix with 50 ul of antibodies... but It is not clear for me If the cell suspension must be adjust to 10^6 in the total 100 ul (So I could mix the antibodies with the double of cells because of the dilution) or taking 50 ul from the 10^6 cell/ml suspension (If I am not wrong, I will have in the tube 500.000 cells in total). Also for the antibodies I do not know exactly how to do because It is said around 0,1-0,5 mg (The point is... how to start?). 

I would like to fix this two things, of course i will have to optimize everything but I need to fix a first protocol to improve it hehe. Soon I will have the cells and I would like how to perfom the trials.

Thanks a lot, really!

Sergio.

Hi Sergio,

the amount of antibody that you mention just seem very high. Have you looked at the company suggested concentrations for this specific Ab? Is this Ab conjugated with a fluorochrome? I normally use 1ul (conjugated Ab) per million of cells. If you have enough sample you should titrate your Ab. 

Erika Lopes

Yes sorry! I meant micro-g hehe.

Yes, I have to check all the antibodies the issue is when I mix them I should have a standard protocol for all of them to make a comparison (I mean, always use the same buffer and the same number of cells etc.).

But with your answer I can have an idea :) (How much is exactly 1 ul in your case).

Thank you!

Sergio