You can see the websites below. It might be helpful for you.

http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=4211

http://www.scienceboard.net/resources/protocols.asp?action=article&protocol_id=27

what cells are you using and what CD markers are you looking at? have a look at antibody data sheet, they always have the staining protocol included in there. Bear in mind that there might be slightly different protocol for different antibody (different company). They also include testing cell line. If you have a possibility to get the control cell line, you can check whether there is something wrong with your staining technique or maybe cell preparation.

When I stain my cells, for example monocytes, use 1-5x10^5 of fresh cells per tube in 100ul staining buffer, add 5-20ul of antibody (depending on antibody; titration might be needed) for 10 min in room temp or 30min at 4C (this should be specified in the data sheet). Then I wash it with 1ml of staining buffer and centrifuge for 5min. Resuspend the pellet in 300ul staining buffer (if I am analyzing the sample same day) or fixation buffer (e.g. cellfix from BD Bioscience, when I am not able to analyzing straight away).

Hope this helps. Good luck

Hi,

Please find below the link to one of my publications which might be useful for your experiment: http://www.biomedcentral.com/1472-6750/12/35

Good luck,

Bianca

Hi Ram, to get an answer for your question you really should give more information. Write, please, following:

1. which cells do you use (are they different from those which you stained before and got a signal?)

2. How do you prepare you samples, why do you think it could harm cells?

3. Which controls do you have, incl. isotype, positive (expressing the marker of interest cells) and negative (cells which do not express marker) controls? Probably, you don't have some of these controls but at least one of them you should have.

4. Which antibodies and staining buffers do you use? Are they different from those which you used before?

And finally, if you have antibodies for desirable marker in different colour AND different clone, and you assume that your cells should be positive (for ex., know it from previous stainings or from literature), you can try them (new/different ABs) and see whether staining works or not.

Good luck!

wash the cells, stain them, incubate in ice for 2o min, wash the cells again, and do you FACS

One easy way to use CD markers in Flow cytometry is to buy the primary antibodies conjugated with fluorophores so you can incubate your cells for 45 min to 1 hour with the antibodies, wash once in PBS before bringing your cells to the flow cytometer. Make sure to include a cell viability fluororescent marker as well.

I think better you share the protocol that you used and did not get the positive population. Give some information about your cell and treatment (if there is any). I think then someone experienced can figure out what could be the reason behind not getting any positive signal.

Thank you for your suggestions.I washed the cells with PBS then incubated with antibody for 30 minutes in dark. Again I washed the cells and resuspended the pellet in 300 micro liter PBS and went for the analysis. After incubation I vortexed does it cause the problem ?

U can try this protocol.

Immunostaining procedure:

Lyse-wash sample preparation method using whole blood:

1. Place 100 ul of whole blood (about 1 million cells) of each subject into 2 FACS tubes.

2. Add 10 ul of conjugated monoclonal antibody (ies) to one tube and isotype control to the other (it is better to vortex the vials of conjugated antibody before adding).

3. Mix and incubate in dark at room temperature for 15 mints.

4. Add 2 ml of BD FACS Lyse (containing < 15% formaldehyde and < 50% diethylene glycol, was diluted 1:10 in deionized water immediately before use) to each tube.

5. Re-incubated in the dark for 12 minutes.

6. Centrifuge at 250g for 5 mints and discard the supernatant.

7. Break the pellet and wash the cells twice by adding 2 ml of sheath fluid, mix, centrifuge and discard supernatant.

8. Re-suspend Cells were in 0.5 ml of sheath fluid with 2% paraformaldehyde.

9. Acquire on flowcytometer.

Hi Ram,

your protocol seems very basic and should work in any case. If you describe the same protocol which you used before (and it worked), then DO NOT change protocol for the staining. In this case, you either have problem with the cells or with the antibodies. To check whether smth is wrong with your cells use an appropriate positive control: the best scenario is to use cells which express the antigen for sure. Alternatively, you can stain compensation beads (for exmpl, from eBioscience) and check whether ABs work in principal. If you have bought new ABs, from the another manufacturer, then you should think about the titration. Maybe, you do not use enough for the staining.

PS: if you will change the staining protocol, you would deal then with an another unknown in the puzzle. And if you add PHA containing buffer, keep in mind that PHA sometimes (and not so rear!) changes confirmation of antigen epitope and you ABs probable would not recognize it anymore. All the time make sure that ABs for the surface markers will work on the fixed cells by staining unfixed cells in parallel at the first time.

Dear Ramit is paramount for you to give some detail. In particular you should explain which type of cells you are using, fixing procedure you are using, even buffer that you use for immunodetection, etc. However, if you think you are damaging cells (but i do not know what do you mean with damage) probably you should work at 4C. So, please, share your protocol.

Hi Ram, Surface staining should be a relatively straight-forward procedure and, as highlighted above, there are many commercial/research protocols online. So my questions would be what tissue are you looking at? And what antigen are you trying to detect? What level of surface expression do you expect - CD4 80k molecules on appropriate T cells while CD25 highly variable dependent on activation status!

Have previously got this assay to work?

As Rakibul says if you give more information it would be easier to identify the problem - tissue preparation, staining protocol or reagent!

If your sample is being damaged in preparation you would see corresponding changes in fsc/ssc scatter.

If your only fluorophore is fitc you can simply include a viability dye either propidium iodide or 7-aminoactinomycin D to identify the amount of tissue damage by the level non-viable cell staining in FL3(~610-690nm).

Defective antibody conjugation from inappropriate storage or bacterial catabolism should be detected quickly using compensation beads.

Once had a user who was given a bottle of water labelled as PBS, in error, who had similar problems until they made up some fresh PBS!

Hi Ram, it is easy to observe whether your cells were damaged. When you do the FSC x SSC plot it is possible to observe the percentage of death cells/debris. Yet you can stain them with Trypan blue just before FACS analysis and observe at the microscope for death cells.

Hi Ram,

as Alessio suggested, it's paramount to explain which type of cells you are using; anyway, that's the protocol I use for several types of cells, not for blood.

a. Harvest cells as you usual do and wash in 1X PBS.

b. Wash again in 1X PBS+ 1% FBS (or 1X PBS+3%BSA), which reduces non-specific antibody binding

c. Meanwhile, count the cells you have

d. Resuspend in 1X PBS + 1% FBS to a final concentration of 200.000 cells/100 µl for each tube

e. Remember to keep a negative control (a tube containing only cells with the isotype controls)

f. Put in a FACS tube the appropriate volume of each conjugated antibody directed to the cell surface marker(s) of interest. You have to do a preliminary test on your antibody, with a negative and an addicted cell line, to choose the right amount.

g. Add 100 µl of the prepared cell suspension (equal to 200000 cells) to each tube.

h. Vortex and incubate for 30 minutes in a covered ice bucket on a seesaw rocker.

i. Wash off the excess antibody in 1X PBS.

j. (Obviously, if you used a primary unconjugated antibody, you have now to incubate cells with the appropriate secondary antibody, for 30 minutes in ice, in the dark and preferably on a seesaw rocker, and then wash twice in 1X PBS).

k. Add 1 µl of DAPI (or a little amount of PI or 7-AAD) to detect alive population.

l. Resuspend pellets in 200 µl of 1X PBS.

n. Analyse by flow cytometry

Remember to exclude debris by measure FSC/SSC, as Valquiria suggest you, and so the doublets (FSC Pulse Width/FSC), and select viable cells (DAPI or PI-).

Good luck and let me know if it works!