Does anyone have experience working with ULA plates? I plan to do a phagocytosis assay with Raw 264.7 and cancer cell lines (adherent). My goal is flow cytometry
I'm wondering about two things:
1. wouldn't these cells need contact (e.g., actin polymerization etc) for macrophages to uptake cancer cells, thus, does it makes sense to have these cells in suspension? (Note: I don't mean NO production etc, just direct uptake)
2. I think this is a slow process. Is that true?
my co-culture conditions could change based on that. I have seen papers use 24, 48 hrs etc but just curious.
Hello Arathi,
I haven't worked with mouse macrophages, but have plenty of experience with human macrophages. Whenever you want to recover the macrophages intact it is highly recommended to use ULA plates. Especially if you are performing flow cytometry assays. If the RAW cells are as sticky as the human macrophages you have nothing to worry about. They adhere well enough to the bottom of the well to differentiate and to perform all their biological functions. Recovering the macrophages for cytometry assessments should be done carefully. Especially if you will be using a viability probe. Retrieve your cells by replacing the medium with cold PBS and gently pipetting them with a p1000.
I hope it helps.
Esmeralda
Esmeralda, Thanks for your reply.
RAWs are sticky to normal culture flasks. To ULA, I don't think they are that sticky. My concern is if this is considered as a good culture setting for RAW cells as by nature, they attach.
Hello Arathi,
Do you have difficulties detaching your macrophages from the regular plates? If phagocytosis is the only assay you are going to determine by FACS, then the use of regular plates should not be a problem. That is if you have no problems with the detachment. If you are including phenotype staining, then the protocol typically used to detach the macrophages is going to interfere severely with your staining. If your protocol for detaching the cells is not that good, then you would not be able to use FACS protocols in macrophages cultured in regular plates. Do not be afraid of using ULA plates. I was skeptical at the beginning too. It is for the best when flow cytometry is concerned.
Best
Esmeralda
Thank you Esmeralda, I'll give a shot with ULA. I tried once before but didn't pursue further. I'll update ya.
Arathi (Sesha) Paluri
I recently tried phagocytosis assay in ULA plate. i got very weird result compared to previous experiment carried out in 6 well plate. I wonder how your experiment came out. i differentiate macrophage from mice BMDM by stimulation with M-CSF. Recently i changed my cell incubator too. so i can not figure out if there is a problem with macrophage differentiation or it is because of ULA plate. Thanks in advance.
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