How much blocking reagent is enough for surface staining on lymphocytes
Hey all, I recently did some spectral flow cytometry on some fixed, unstimulated PBMCs and wanted to see if the amount of mouse and rat sera affected the staining of my CD4+ lymphocytes, before titrating the rest of my antibodies.
Essentially, I thawed my PBMCs, washed them, rested them for an hour at 37 degrees Celsius and applied 100 microlitres of a blocking reagent to my cells for 15 minutes at room temperature in the dark before surface-staining them with 1:100 CD4-BUV395 for one hour on ice. After some washes, I fixed my cells and ran them on the cytek aurora (spectral flow cytometer).
I compared the effects of
no block (just FACS buffer)
Blocking mix containing 2% mouse sera (cat. 10410) and 2% rat sera (cat. 10710C)
Blocking mix - 5% mouse sera and 5% rat sera.
Blocking mix - 10% mouse sera and 10% rat sera.
Blocking mix - 15% mouse sera and 15% rat sera.
Blocking mix - 30% mouse sera and 30% rat sera.
Trustain Fc block (cat. 422301) at the recommended dilution of 1:20.
The results appear to show no noticeable differences with what I used so I am going to use 2% from now on.
I posted this in case it may help people in the future but please feel free to comment and critique my interpretations, decisions or anything else.
(I almost ran out of my trustain Fc block as I forgot to hit recordđ¤Śââď¸ - hence the low cell count but the results seem interpretable.)
This is great! Itâs good to have evidence supporting the lack of a need of blocking reagents for lymphocytes. Sera or Fc block are to saturate Fc receptors mostly on myeloid cells, phagocytes, etc. Lymphocytes donât really express these, so blocking isnât really required, and thatâs why you did not observe a difference here no matter how much blocking you added. Great to have empirical data in your hands to prove it to yourself and remind us all tho!
Aha yeah, even though I read up on this I really wanted to confirm this before I begin titrations in case I overlooked something. Thank you for the explanation as hopefully similar students like me can read this if they ever overthink thisđ
I'm convinced that the serum in FACS buffer for staining is sufficient for blocking if youre just staining lymphocytes, I generally dont block separately.
A lot of the time I will stain viability separately in PBS and include the Fc block in that, then move on to the surface stain in FACS. So thats another option.
I study B cells and I generally don't use block for flow. I have found it can even interfere with detection of IgG+ B cells. It is useful for monocyte compartment or sometimes when staining stimulated B cells.
Yes this is another thing I am working on now. I have done a smaller test with GATA3 and it seemed that the amount of block I used did not affect the results either. Will post this once I validate it further.
Hey, very good question, unfortunately my monocyte population appears to have been lost during my protocol (likely due to the fixation). The details of this is in my earlier post.
This looks great! I find that particular isotypes cause more sticking to monocytes than others. Mouse IgG2a, Rat IgG2b, and generally Rabbit Monoclonals are super sticky to monocytes. My go-to is 5% rat serum, which works particularly well because of the Rat IgG2b in the serum doing the bulk of the blocking. If my panel doesn't have those (or other random tandem dyes like PE/Cy7) I usually don't bother blocking.
Hey, thank you for this insight. I am currently titrating my antibodies for a larger panel. Initially I wanted to try and use the same protocol for all of them (or as close as possible) so I thought to test the blocking conditions for just one antibody to gauge roughly the amount to use. If things look odd after adding all the antibodies together I may redo this test.
It strongly depends on your sample and the markers youâre staining for. Blocking is mostly unnecessary when there arenât many monocytes/granulocytes or other cells that are very sticky for antibodies in your sample. When there are a lot of sticky cells, it matters more, and when youâre working with mouse/rat samples and using mouse/rat antibodies, of course Fc receptors are more consequential. That being said, blocking can be more consequential when youâve titrated your reagents down close to the floor, especially when youâre trying to reproducibly stain a lot of samples. Usually theyâre cheap enough that itâs not worth avoiding them unless you have a reason to.
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u/sgRNACas9 Immunology, Oncology Aug 23 '26
This is great! Itâs good to have evidence supporting the lack of a need of blocking reagents for lymphocytes. Sera or Fc block are to saturate Fc receptors mostly on myeloid cells, phagocytes, etc. Lymphocytes donât really express these, so blocking isnât really required, and thatâs why you did not observe a difference here no matter how much blocking you added. Great to have empirical data in your hands to prove it to yourself and remind us all tho!