I previously used APC-PDGFRa (clone APAA5),eBioscience for FACS. At that time dilution, 1:50, and 1:100 worked very well. but nowadays it's not working. could anyone help to guide how it happens and why I can't see the PDGFRa+ population? In my opinion, I purchased new Ab with the same catalog but a different lot. so it has any impact on the quality of FACS Ab. or please suggest a good antibody to detect the PDGFRa+ population by flow cytometry.
thanks.
If anyone had used different antibody then pls share clone and catalog. thanks
Did you test the same reference sample (ie. a cell line) which definitely includes PDGFRa+ cells? Were the staining and acquiring conditions the same (temperature, time, cytometer, settings etc..)? Did you include other antibodies in your stain? Does the antibody bind compensation beads (ie. BD comp beads)?
If everything you did is technically sound, you should contact the company and ask for another sample from a different lot (at not added cost..) to test if the problem is batch-specific.
You can have a look on the following link:
https://www.thermofisher.com/antibody/primary/query/PDGFRA+(CD140a)?gclid=EAIaIQobChMIsvW2v4SM6QIVBiQrCh0ERgrCEAAYASAAEgJJv_D_BwE&s_kwcid=AL!3652!3!423082964381!e!!g!!pdgfra%20antibody&s_kwcid=AL!3652!3!423082964381!e!!g!!pdgfra%20antibody%3Fcid%3Dbid_pca_aup_ro4_co_cp1359_pjt0000_bid00000_0se_gaw_nt_pur_con&ef_id=EAIaIQobChMIsvW2v4SM6QIVBiQrCh0ERgrCEAAYASAAEgJJv_D_BwE:G:s
I have used the same clone as you but from Biolegend, they tend to work very good and they are quite cheap. Ref: 135907. Although I have noticed some differences within the staining, my case is tumour associated fibroblasts, but that depend much on the batch effect of the sample processing.
Gabriele Casirati thanks for the answer. Yes, all the conditions were the same and the same mice tissue was used. the combination of antibodies was also the same. I agree with you about the compensation issue. But I could not detect single stain positive results. e.g if the expression of PDGFRa+ cells usually between 20-40 % in lineage negative fractions but using this antibody only 5-10%. I think this antibody is less efficient. I will try other options as you suggested.
Francisco Javier Rodríguez Baena thanks for the answer. I agree with you. i will try that one next time.
I will see Nap northern Ben
and Anju Kaushalthanks for the information. Have you ever used this Ab and what was the result? how many percent population was positive for PDGFRa.
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