Hi everyone,
I would like to do a phagocytosis assay with my selected compound using RAW 264.7 cells. I want to know in what cell culture plate I should be doing this experiment - 6 well, 24 well or 96 well plate and also what is the seeding density I should be starting with?
Thank you.
Hi,
it mostly depends on what you will use as a read out for your experiment. If you are planning to do microscopy with several concentrations and several time point then a glass-bottom 96 wells plate is the way to go. I you gonna used spectro-fluorometer then a black plastic bottom 96 wells plate will do. If you want to isolate RNA then 6 well is probably better though you can isolate RNA from 96 well plate with special kit (that are pretty expensive). Raw macrophages divide so you should plate in order to obtain 80-90 confluency the day of read out.
Hope this help.
Best,
Alexandre
As is said it depends on what type of experiments you are going to perform, in our lab we use 96 well plate - we use spectrophotometric methods.
Thank you all for your insights!
I actually want to test my compound's effect on the phagocytosis in RAW Cells. I referred some articles but, many of them were using kit based method and also in a 96 well plate using Fluorescence microscopy ... is it possible to do without kit with fluoroscence microscopy ...? :(
Hi,
you have to tell us what the kit you are talking about.
I think you can do it with microscopy. Just make sure that you use glass-bottom black 96 well plate and that your raw cells are not too confluent the day of experiment (too confluent will make the analysis more complex). You can also use confocal dishes if you do not have too many different conditions.
How do you plan to measure phagocytosis ? with fluorescent beads ? Do you plan to add other makers (EEA1 for early endosomes; LAMP1 for late endosomes-lysosomes).
If you plan to do it in live cells I recommend you to choose a limited amount of conditions. You will have enough time to image these condition in a reasonable time (there will be comparable). Make sure to acquire a lot of cells for each condition, this will facilitate statistics and conclusion.
Best,
Alexandre
Hi Mr. Gidon!
We have actually Phagocytosis Assay Kit (IgG FITC) from cayman chemical.
I am attaching the Manufacturer's manual.
Hi Saikrishna, you can call me Alexandre !
if I understand correctly you are using IgG-FITC to monitor endocytocis. You will have to use a 488 nm laser line for FITC excitation and collect emission using "GFP like" setting (i.e. collection between 505 and 550 for eg).
The manufacturer's protocol is pretty well detailed you can follow it safely !
Best,
Alexandre
A cheap homemade method that does not require a kit or fluorescence was published by Anand et al. J. Leukocyte Biol. (2007) 82:1257. It uses opsonized SRBC and a hypotonic wash to lyse SRBC that are only adherent. Phagocytosed SRBC are then counted using only phase contrast. In this paper cells were grown on glass coverslips, but it can be adapted.
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