Currently, I am doing a research on NKG2D ligand expression on cancer cells.
I would like to use dual color staining, which are APC-conjugated Ab for ULBP-1 and PE-conjugated Ab for ulbp-2.
When I do it in single color, the ratio is 1:5 (ab:PBS), therefore I stick to this ratio when I prepare my antibody cocktail: 5ul APC-conjugated + 5ul PE-conjugated + 20ul PBS to one sample.
The results are weird when compared to single color. For instance, ulbp-2 expression has reduced in dual color setting compare to PE-conjugated anti-ULBP2 alone.
What is the problem?
each time you run new experiment , go for single tube stained Compensation. this will help you reducing the overlapping and thereby better display. also, make your cocktail in 100ul pbs and mix well. Do it in dark room and keep for 15 min incubation in dark. You can also try keeping the stained tube in ice.
anyways , maintain a record of the voltage and compensation set up of each experiment separately.this will help you to recheck the set up and you can change settings and go for better display too.
you can do titration of antibody to find out which concentration best suits your assay. but do change settings first and observe the results. later you can do titration and also other conditions like staining on ice, incubation at 4 degree C and so on
APC and PE should not have compensation problems. I agree with Greeshma, try to make compensation controls. Also try to check the literature to see if these antibodies don't have cross-reactivity. For cell satining you should be using PBS + 2% FBS.
FBS help to keep cell viability. I don't heat inactivate my FBS. You will find in the literature a lot of controversial articles about heat inactivated FBS or not.
I agree with the above comments on compensation. There should not be a problem of compensation when you're only using PE and APC. Are you using an Fc block? As you're not interested in an Fc receptor, I think it would be beneficial to use an Fc block before your staining to reduce the unspecific binding of your Ab to Fc receptors. As important as having single colour controls, it is important to have an unstained control to set your voltages correctly. Using your unstained sample, you can correct the voltages for each channel and put all your values to zero (around) so you know whatever signal you get from your stained samples will be due to the staining. Good luck!
PE and APC are two large fluorophores (~240KDa), I don't know the distribution of your two proteins of interest but if they are very close to one another, maybe you see decreased signal due to steric hindrance. Do you see the same decrese in signal when using two stains simultaneously if you use other smaller fluorophores ?
FITC being ~400Daltons, it's much smaller than PE and is worth trying. Do you know if FRET experiment have been done between your two molecules of interest, to know if they are (or not) close to one another ?
FRET is a fluorescence method used to know, for instance, if two molecules are close to one another. You excite one antibody coupled to a fluorochrome at a given wavelength (which should not excite the fluorochrome on the second antibody targeted at the other molecule of interest), the second fluorochrome should be excitable at the emission wavelength of the first fluorochrome. As this energy transfer is efficient only on very short distance, if the two molecules are close to one another, exciting the first fluorochrome will results in it's emission transfered to excite the second one. This will translate into a measurment of the emission of the second fluorochrome, and no (or very little) emission from the first fluorochrome. If they are distant enough, then you will only measure the emission of the first fluorochrome and nothing from the second fluorochrome...
For some general information regarding FRET and some litterature, check :
http://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer
Attention, it is the FINAL dilution of your antibody that counts. If what you call "sample" is the same identical volume all the time, then you're OK. By the way, from what you described, you actually dilute your antibody 1:6 (in your staining cocktail). Did you titrate these antibodies individually ?!
HTH
Hi Kee,
When fluorescence signals appear differently in a fully stained sample compared to the single stains, this could be due to a number of reasons. These are related to the interaction of the antibodies or fluorophores. One of these is to do with antibodies binding to each other due to binding of PEG. However, this only really occurs with some Brilliant Violet reagents, and so is not relevant here.
FRET
As I understand it, FRET typically applies to the photo-active molecules within the overall structure of the fluorophore (or tandem), and so doesn't often apply to individual fluorophores that are close to each other.
STERIC HINDERANCE
You might be looking at steric hinderance, which Frederic brought up above. I suspect this is the cause.. This might occur if your markers of interest, ulbp-1 and -2, are located very close to one another on the cell, or are part of the same structure on the cell. Essentially, both antibodies cannot fit in the space required for both to bind efficiently, and so they compete. This will result in one fluorophore appearing higher than the other, or both appearing reduced, compared with single stain controls.
The trick with steric hinderance, is that it will only appear in fully stained samples, and obviously will not occur in single stain samples (as there is only one antibody present).
The way to test for this, is to try binding the antibodies separately one at a time on the same sample (stain ulbp-1, wash, then stain -2; and on a separate sample stain ulbp-2, wash, then -1). This way, you can see if the staining order makes a difference to the fluorescent signals compared to the normal fully stained sample.
POOR ANTIBODY MIXING
If the antibodies arent mixed properly in the buffer before being added to cells, you can get heterogenous staining of cells.
As Thomas noted, there are some requiered conditions for FRET to be a reason of signal decrease. The two fluorescent molecules must be close to one another, the combination of used fluorochromes must be compatible with FRET (not all fluorophore will emit at the requiered wavlength to excite the second one (PE and APC are known to be used in FRET experiments). Of course steric hindrance and FRET are not mutualy exclusive, and your strange behaviour can be a result of those two phenomena (or not).
As Thomas mentionned, it's also a very nice approach to test the two steps staining, if only the signal of the second staining decrease compared to single stains, then steric hindrance may be the answer. To rule out FRET, depending on the FACS available, if you can shut down the 633nm excitation source you have and still measure an APC signal when exciting your cell with a 488nm (->excite PE -> FRET -> APC signal), then you have your answer. Those very simple experiments should most likely give you more information regarding your observations !
Good luck. Keep us posted !
Tyring be a cheapskate. Anyone made their own brilliant violet staining buffer? Stain with extra PEG?
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