I have been working with a protocol that uses a wash and binding buffer with 0.05% tween20 for flow cytometry. Recently I switched to using MACs bead for my secondary, but they don't have tween in their wash and staining buffer. I'm wondering if it is bad that I have tween or if it is helping me with non-specific binding as my past training has told me?
Currently I have intracellular antibody targets, but I am testing cell surface markers and I see no staining. Maybe tween works well for my intracellular targets but not cell surface?
Hi Becky,
For surface staining flow cytometry, I never use tween 20 in any of the solution. But, tween 20 has been used in intracellular staining flow cytometry where it can act as permeabilizer (0.5% in PBS) following cell fixation.
Are you attempting to do combined surface and intracellular staining for flow cytometry?
If this is the case, 0.1 % saponin-PBS (fixed with 4% paraformaldehyde/PBS) is a better option compared to tween 20 which is harsh. Normally, I do surface followed by intracellular staining. But, some flurorochromes are sensitive to quenching by fixation step, depending on the fixative agent you used. In that case, I do intracellular followed by surface staining.
Do you know if samples diluted with 0.05% tween-20 interfere in CBA cytometer readings?
Hi Camila, when you say CBA cytometer, do you mean a bead array cytometer? I have not had any experience with tween getting in the way, I used tween-20 in a Luminex assay and it helped keep the beads from sticking to each other. I would check with the CBA cytometer handbook to make sure tween-20 is ok.
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