Hi, It is better not to use the beads, but use your actual samples (tested cells) if applicable singly stained for each dye you are using, and have another sample containing all dyes... this is the best way to set a template for future samples acquisition and analysis. another suggestion is to change the antibody (company or clone) or go again through your procedure (working steps) to see what went wrong. Also you might need to check the calibration of the cyclometer you are using. Thanks

Hi, several to be checked before throwing away yours Abs.

1) are you sure your sample expresses the Ag you are monitoring?

2) if yes, Have you performed dose assay with your Abs in order to chose the best working concentration.

Following up Ibrahim's comment, use your sample instead of beads if you can (always better for autofluoresence for example) rather than beads.

if dealing with a multicellular population with cells having significant autofluorescence and size differences (alveolar fluid for examples), use beads to create your compensation board.

Alexis

hi alex and ibrahim, i did individual staining multiple staining and checked dose assay. it works fine

but for multiple staining when i set the mechine by compensation beads using PMT voltage, for sample its not showing the possitive population. i am thinking it may be related to compensation.

Hi Komal, could you give us a little bit more information on the type of cells you are running, the antibodies and the cytometer configuration ? It will be easier to help..

Dear Hugo :leukemic cell line, surface markers CD34, CD38, CD 45. Canto mechine,

Hi Komal,

What did you make your FMO with and how did you set up the voltages?

while compensating two distinct populations( negative and positive) should be there.Otherwise you can't compensate. If you are using a homogeneous population you might see variation in expression of marker but it could not be positive or negative.

In that case you can stain cells and then mix unstained cellls in the compensation tube at the time of acquisition.

As Raphaelle stated, we need a lot of information to help as there are numerous theories about what can go wrong with a multicolor flow cytometry experiment. I personally do 8*8-color labellings on my hMSCs and compensate with beads but all fluorochromes are not suitable for this method. I guess you are using compBeads do you manage to get your 50%pos/50%neg bead populations ? Also, I know Diva does the compensation automatically, do you compensate this way or later-on ? Do you analyze with diva or flow-jo ?

hi Hugo, asif , raphel.

yes i can see both possitive and negative population 30% negative and 80% possitive for percp cy5, cy7. for PE i cannot see negative population even after mix with unstained cells during aquicition. i dont no why.

i do FMOs that is to gate negative population example for FITC a tube with cells+PE+CY5+CY7 accept fitc so i can gate negative for fitc.

first i run unstained, then individual beads with flurochorme conjugated antibody, i am using FITC, PE, Cy5, Cy7, BV and set for possitve population. after compensation i use FMOs to exlude one population that is florochrome minus 1. but the problem is after setting the gating using compensation and fmos when i run samples i cannot see the possitive signals.

i play with voltage , when i run PeCy5 it usally bleeds in to PE, pecy7 i try to reduce the voltage for Pecy7 and PE. but when i run PE it bleeds into pecy7 and again i reduce votlage for pecy7 , this is actually causing the problem. can u tell me how to overcome this problem

I will suggest you to just adjust voltage in your unstained cells then run your all samples (Compensation and test tubes) . then compensate samples by using third party software like Flowjo

Oh ok but i am trying to compensate on the mechine they actually spill in to each other channel.. If i reduce voltage of pecy5 for pecy7 then run pe again it spill in to pecy5 if still reduce the voltage they dont give signal for sample

As asif says, you should set the voltages with unstained cells only and deal with compensations during the analysis, with flowjo for example.

When setting your voltages with unstained cells, you should have your population contained between 10^0 and 10^1, as much as possible. Then you have to titrate your antibodies with your cells of interest and choose the concentration that allows you to have a positive population which can be clearly distinguished from the unstained population.

Keep in mind that for some markers you might only achieve shifts rather than a true distinct population (to make sure that this is truly due to a specific binding and not bleeding of some other fluorochrome you need the FMO, but I think you got that point). An example is Bcl2 which is expressed in all cells and for which one might look at the level of expression (then looking at the total MFI).

One more thing, in some situations (activated PBMC vs normal PBMC for example) the cells have greater autofluorescence, so make sure you're using the right cells for your "unstained control".

To summarize, do as so: adjust your voltages with unstained cells, acquire all your samples and compensation controls (beads or cells, I personally actually like beads as they give a strong positive population), record your raw data like this, analyze it with flow jo, and let us know if you still can't see your positive populations.

I hope it'll help. Good luck

I guess you are trying to hard with the compensation before running the actual sample. I would suggest: Forget about everything, do unstained cells and single stainings additionally to your fully stained sample. Set the voltages according to the single stainings (that it looks as good as possible).

Try to bring the PMT voltages of PE and Percp-colors closely together, that will ease up the compensation later. Dont do any compensation yet and forget about beads in the beginning.

Try to analyze your samples with flowjo then and try to do a compensation in flowjo with your single stainings. If you still run into problems, leave me a message, I can have a look at your fcs 3.0 files (dont use 2.0 because of the compensation issue).

Good luck with the stainings ;)