I'm working on CD34-FITC and used CD34- isotype control antibodies from miltenybiotec company, but the CD34-FITC control is not available in our lab right now but I have CD86-FITC isotype control from miltenibiotec company.(thus both of isotype controls from miltenybiotec).
So, can I use CD86-FITC isotype control instead of CD34-FITC isotype control?
I imagine you are speaking of "istype controls" (?)
If you have a look to commercial catalogs, there is generally only one isotype control per fluorochrome (e.g. FITC, PE etc...) and per isotype (obviously) in any catalog even if there are hundreds of specific anti-CDxx FITC-conjugates. This means one isotype control is generally used for many different specific MAbs-FITC. Indeed, this is a problem since FITC reagnets may have very different concentrations and F:P ratio, thus one control is rarely the perfect match, especially in the case of no-wash protocols. But for your question, I would answer: Yes. You would then do the same as most other people generally do, using one isotype conjugate for several different FITC-MAbs. I would add: try to get the information of Ab concentration for both the specific MAb(s) and the isotype control and best use similar doses (if possible).
But still, you may end-up with differences in the F:P ratios (rarely indicated for commercial reagents).
For that reason, many "flowers" have suggested the use of fmo (Fluorescence minus one) strategy. You can follow forums on that FMO strategy e.g. on the Purdue University FCM list (ask Google for FMO & Purdue). This means all other color conjugates except the one you need to control. Whereas I agree that this may be acceptable (and cheaper) when using stain & wash protocols, it would surely be disputable in case of no-wash protocols.
Hope that helps
I imagine you are speaking of "istype controls" (?)
If you have a look to commercial catalogs, there is generally only one isotype control per fluorochrome (e.g. FITC, PE etc...) and per isotype (obviously) in any catalog even if there are hundreds of specific anti-CDxx FITC-conjugates. This means one isotype control is generally used for many different specific MAbs-FITC. Indeed, this is a problem since FITC reagnets may have very different concentrations and F:P ratio, thus one control is rarely the perfect match, especially in the case of no-wash protocols. But for your question, I would answer: Yes. You would then do the same as most other people generally do, using one isotype conjugate for several different FITC-MAbs. I would add: try to get the information of Ab concentration for both the specific MAb(s) and the isotype control and best use similar doses (if possible).
But still, you may end-up with differences in the F:P ratios (rarely indicated for commercial reagents).
For that reason, many "flowers" have suggested the use of fmo (Fluorescence minus one) strategy. You can follow forums on that FMO strategy e.g. on the Purdue University FCM list (ask Google for FMO & Purdue). This means all other color conjugates except the one you need to control. Whereas I agree that this may be acceptable (and cheaper) when using stain & wash protocols, it would surely be disputable in case of no-wash protocols.
Hope that helps
Dear Philippe Poncelet
thanks for your helpful and kindly respond
exactly my mean is isotype controls and i edited my question...
actually the isotype controls that i said purchased from miltenyibiotec company, thus their concentration and protocols are the same.
Hence, if i use isotype control instead of another isotype from same company with same fluorochrome, is it work ?
You have to make sure that your CD34 and CD86 antibodies are from the same host and same isotype (e.g. IgG1, IgM, IgD etc), then you can use the same control antibody for both assays.
In routine practice, I fully agree with Korcan's answer. If you really need to study specific staining in details, (and if you can affort it) full specific inhibition with pre-incubating the same MAb at 10x to 50x concentration before coming with the FITC conjugate would be most appropriate. But one generally can/will not do that (expensive, cumbersome ...).
For newly appearing discussion on the search for optimal negative controls and isotypes in (difficult) no-wash procedures such as those applied to Microparticle analysis, see: Inglis et al, Cytometry A, online issues. PP
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