I have Pe-CD4 antibody that I need to titrate, however, the cells that i have are a CD4+ cell line. this is a problem because all my cells are staining CD4+ with no negative cell population, thus i cant get my staining index. So, I bought some isotope controls that bind to the the PE antibody, but i dont know how that will help me out. is there a way i can get away with this?
Make a sample with a 50:50 mix of cells expressing a CD4 - cell line and your CD4+ cell line.Take an aliquot for unstained control. Stain aliquots with CD4 at different titrations.
Run unstained control and set the markers to define the unstained peak. Run the various titrations through the machine, if the negative peak starts to move upscale then your cells are over-stained and this concentration should not be considered as the correct concentration of antibody. (Note: this is why you need to have a negative peak in your stained sample, otherwise you will not see the impact of overstaining).
Usually the sample with the highest ratio of positive signal to negative signal is considered accurate - but if you can distinguish a resolved peak away from the negative with lower ratios those concentrations may also be ok to use.
The isotype control is not a negative control and should only give you info from non-specific binding, it is not helpful in defining an ab titration.
Use unstained cells, isotype control cells and CD4+ antibody labelled cells. Compare cell population scattering and mark isotype control as negative population. Rest of population will be your CD4+ cells.
For antibody titration, have to check by using range of concentration; first have to look for concentration suggested by the manufacturer's datasheet.
You mean mix the cells (stained and unstained) with the isotype? or isotype with each (stained and unstained cd4)?
No, you need to acquire both the sample separately.
Although if you are using isotype control, there will be no need of unstained.
The isotype control will be your CD4 negative population.
Make a sample with a 50:50 mix of cells expressing a CD4 - cell line and your CD4+ cell line.Take an aliquot for unstained control. Stain aliquots with CD4 at different titrations.
Run unstained control and set the markers to define the unstained peak. Run the various titrations through the machine, if the negative peak starts to move upscale then your cells are over-stained and this concentration should not be considered as the correct concentration of antibody. (Note: this is why you need to have a negative peak in your stained sample, otherwise you will not see the impact of overstaining).
Usually the sample with the highest ratio of positive signal to negative signal is considered accurate - but if you can distinguish a resolved peak away from the negative with lower ratios those concentrations may also be ok to use.
The isotype control is not a negative control and should only give you info from non-specific binding, it is not helpful in defining an ab titration.
Use of isotype controls is deprecate in flow cytometry. Please stop this bad habit. The non-specific binding of isotype controls is different from the non-specific binding of your antibodies of interest, and therefore the background fluorescent signal that you get from using isotype controls is quite meaningless.
Godfrey,
this is an interesting question! Titration examples always involve a population of positive and negative subsets of cells to calculate the signal to noise signal. I run autofluorescence for determining the noise and another tube with the conjugated antibody. I really don’t know what could be the best option. The use of isotype control must be considered with careful because there are lots of details to analyze (protein fluorochrome ratio..).
Ann Atzberger,
Your idea is to mix two different cell lines. One positive and one negative cell line for CD4. How about the autofluorescence? Different cells have different autofluorescence that could interfere with the result. Am I wrong?
Hi Flavia,
absolutely it is better to have matching autofluorescence, however if the positive signal is well resolved it will not matter too much. If the positive signal is low then it would be necessary. The isotype is irrelevant and would mask the overstaining of the isotype and the overstaining of the test sample. Raffaello's point above is valid however not all scientists/reviewers are on board with it; all the more reason to understand what an isotype control is and where it can be used in a meaningful way. You will find loads of info on the Purdue cytometry forum.
Raffaello Cimbro Sorry, but I do not believe the aim of isotype control is to verify non-specific binding, instead it is primarilly used to verify deposition, entry and and capture in cytoplasm and/or nucleus , (in case of intracellular staining that cells are permeabilized) ,and mainly to evaluate capture antibody for Fc receptors in some cell types, as majority of immune cells, that can generate false-positive results , even though there is no antibody-antigen binding . I have tested different Fc blocking agents in my cells (human monocytes and DCs), and comparing a good one (ex: 0,5% non-fat milk) with a bad one or even without blocking step that were incubated with a isotype control (that do not bind with a non-specific target, but its Fc portion can be captured by Fc receptors of these cells or can be captured inside the cell) give me very different results: using non-fat milk have genrated 3% of postive cells whereas with bad blocking agents or without blocking have generated a mean of 97% of positive cells
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