Which antigen or antigens are you assessing? Are you intending on activating the platelets? What was your dilution range?

It's difficult - no, impossible - to titrate an antibody without a negative population. By increasing the antibody concentration you will obviously increase the staining - in a linear or otherwise fashion.

BUT!! You also increase the "noise" in the set up which you won't see without a negative control.

My suggestion is to do your titration again with 100 X 10^3/ul in 100 ul but also additionally stain with the same dilutions of its matched isotype control in separate tubes i.e. one with the same fluorophore with an identical fluorophore to antibody stoichiometry. That way you will now see the "noise" in the system with the increasing concentrations of the matched control antibody. You can then plot your antibody concentration but this time against the calculated positive MFI to negative MFI ratio values and the plateau gives you the optimal dilution value.

However, if your antibody comes with a data sheet that tells you it's in that ball park for the working dilution and volumes then it's not usually too much of a worry.

NB If this is CD marker that increases with activation (e.g. Gp IIb [CD41] or Gp IIIa [CD61]) then you'll need to stimulate the platelets with a suitable agonist as well because the signal will increase by exposure of alpha-granular receptors.

hope this is of help and good luck with your study.

Hi,

what company the antibodies have been purchased form?

Greetings,

KB

Hi Zaianb,

Commercial antibodies usually work well over a range of cell concentrations. If you have 100x103 platelets/µl, that is 1x105 platelets/µl, and in 100µl you have 1x107 platelets. The antibody instructions suggest you use 1x106 cells in 100µl. But that is probably OK. Platelets are much smaller than most human cells, and have fewer antibody binding sites. 

Your titration results suggest there is a wide range of antibody concentrations you can use. Pick a concentration that gives a good signal, and will work well with other antibodies (if you are doing a multi-colour experiment). 

It is important to be able to distinguish between the levels of your marker in the samples you wish to test. If you are measuring platelet activation, make some activated and resting platelets, and verify you can measure the difference between them. 

You should test if it affects your results, but it is good to fix your samples after labelling - you may be able to store them, ship them, and analyse them at different times after preparation. If you are labelling 100µl of washed platelets, you can fix with 900µl of 1% paraformaldehyde. 

James

Thanks Dr.Barry Richard Wilbourn for your replay

I assess the Platelet CDs markers (CD42b, CD49b, CD41a, and CD61) in resting state.

I have already run isotype controls along with antibodies titration, but I didn't include it  when I plot the curve.

- Could you please tell me what do you mean by calculated positive MFI?

Is it different from MFI that is given by the machine?

- Can I find a solution for the data that I have for the samples that I was run without cell counting adjustment?

I appreciate your help

 Thanks Mr.James Mcredmond

I appreciate your help

Hi Dr.Katarzyna Bednarska

I have been purchased form BD company

Sorry. I didn't phrase that very well. I meant that you calculate the ratios of the MFIs of the positively stained platelets to the MFIs of the platelets stained with the isotype control for each antibody concentration. The isotype control values are the "noise" and the positively stained are the "signal". You'll observe that both sets of MFIs increase with antibody concentration but they'll be a maximum ratio for the optimum concentration i.e. the maximum signal to noise ratio. I hope this clarifies your question.  

Dear Dr. Zaianb Al-Najjar,

Based on the experience of my colleagues, specialists of hemostasis, I recomend the Becton Dickinson Procedures for staining of blood platelets:

http://www.bdbiosciences.com/ds/is/tds/23-3371.pdf

Some suggestions: Avoid labeling of 100 microliters of blood (or PRP); it is 20-fold excess of blood volume in relation to the concentration of antibody. Use 20 microliters of antibody to 5 microliters of blood (or PRP). Incubate mixture for 20 minutes at room temperature in the dark. PFA fixation may also take place at room temperature (50-60 min).

Best regards,

Katarzyna

... let me quote BD: "NOTE: 100 μL of whole blood yields enough fixed blood for 20 tests. If additional fixed blood is required, prepare the appropriate number of tubes. Do not increase the volume of the blood or paraformaldehyde in the tube as this will increase the possibility of platelet aggregation." 

http://www.bdbiosciences.com/sg/resources/protocols/platelet_activation.jsp

Hello,

Generally we adjust the zero (background noise) on diluted platelets and labeled with isotype control corresponding to the isotype of the anti-CD. For reading, performed after labeling with anti-CD, we keep the same conditions and zero compensation.

you must also test platelets reports / Anti-CDs at several dilutions to find the optimal dilution in its own working conditions (cytometer, antibodies ...) which gives the best results with the minimum of antibodies.

best regards

The procedure published by Becton Dickinson, quoted by me in the previous post, is designed so that you do not need to adjust the amount of antibodies or dilution of platelet suspension. 

Best regards, KB

Dear Zaianb Al-Najjar,

If you want to make measurements for resting platelets, I suggest that you use whole blood samples with no nwash/lyse method instead of PRP.  Also, you need to collect blood sample with special attention to prevent activation by blood drawing method. You need to fixate the 50 uL whole blood with 2% PFA (Paraformaldehyde) solution, before your staining. I suggest that you read the articles by Alan D. Michelson to set up your study in light of previous studies. I also recommend his book, Platelets. One article by his group is "Application of flow cytometry to platelet disorders. Linden MD, Frelinger AL 3rd, Barnard MR, Przyklenk K, Furman MI, Michelson AD Semin Thromb Hemost. 2004 Oct; 30(5):501-11." For monoclonal antibody titration, you can look up to Mario Roderer's pages on the web. 

Thanks to all

I appreciate your help and efforts

I want to make sure that:

if I measured the expression of platelet CDs marker in resting platelet by using 100 ul of washed  PRP without adjusting the platelet count on 100X103 and stained with 10ul of conjugated monoclonal antibodies, is the MFI results reflect the real expression?

If not, is there any equation to adjust to get the exact MFI?  

Would be better to follow BectonDickinson protocol: use 5 ul WB, PRP or isolated platelets (3 x 10exp8/ml) and 20 ul of BectonDickinson antibodies. Five microliters of PRP, not hundred microlitres. Otherwise you will get false results.