sc_queen, on 09 January 2012 - 10:35 PM, said:
I will be titrating my FACS antibodies (and this is my first time). Can anyone please answer some very basic questions below?
- Which cells should (or can) I use to titrate? (I will be sorting primary cells from normal tissue and/or normal epithelial cell line. Can I use ANY cells?)
- Is there any case where Fc-blocker is not required?
- Does the procedure somewhat vary depending on conjugation status (flourochrome, biotin, or non-conjugated), and if so, why?
- Do you recommend serial dilution or adding different amount of antibody to the same volume of buffer?
1. You can use any cell you want to do a titration but you have to ask yourself what are you optimising for. If you are detecting say, marker A on a specific cell line B, then the wise thing would be to use cell line B + antibody against marker A. However,
this is only the case if you know cell line B expressed marker A (this can be done by literature search, gene expression experiments, immunostaining etc.). The flow antibody spec sheet usually comes with a 'positive control cell line' which will definitely express your marker of interest (it is usually immortalised cell lines or isolated mammalian cells).
2. Fc blocker is usually used on mouse/human serum analysis
where you test for immune cell subpopulations. The rationale is that secondary antibodies could potentially bind to primary cells hence giving a false positive background. The main thing is to have a matched isotype control (this is the Ig of your primary antibody - again can be found in the spec sheet). It is advisable to use the same concentration as your primary and then use this as the 'background reference'.
3. If you're using the primary antibody for the first time, I would recommend you titrate the primary and secondary. Although most of the time, small changes to the secondary dilution doesn't affect the final data much (but who knows, don't risk it
4. This is totally up to you. I normally just add the appropriate volume in the flow tube. E.g. 0.1, 0.5, 1.0, 2.0, 4.0 ul into 100ul cell suspension. I find this easier to handle if you have alot of tubes.