I have recently graduated and taken a new job starting up a tissue culture/efficacy testing lab at a personal care company. I've been trained as an immunologist so my go to has been FACS but I do not have that equipment in my new lab.
I want to look at cell adhesion molecules but need a new way to do it. I have a BioTek microplate reader that can do all kinds of things. I'm thinking I could possibly prep my cells as if I was doing a FACS experiment, plate them in a 96 well plate, and read the fluorescence on the microplate reader.
Does anyone have any suggestions on the above idea or other types of assays I could do?
If your antibody recognize the extracellular part of your surface proteins, you should proceed using standard immunofluorescence without permebilizing the cells.
For the analysis, there are published paper quantifying the intensity of fluorescence on from multiwells using the Odyssey scan (this has been used to quantify the cannabinoid receptor, Mackie 's lab).
For a standard microplate reader the main issues are the sensitivity of the instrument and the amount of background when working with microplates (Odyssey solves this problem using infrared fluorescence).
The easiest, fastest and cheapest thing to do would be microscopy, whether conventional or fluorescence. You can count the number of positive cells and assign an arbitrary scale for the intensity of the reaction (like 0 vs +++). I would be hesitant to read the OD from whole cells from an elisa reader. It might be prone to artifacts due to the 3-D structure of the cell, uneven receptor distribution, elevated background OD. If you need more precise quantiation of receptor, you may need to extract membrane proteins and attach them to your elisa wells and run your EIA or run then on a gel and stain.
Find a university nearby that has flow core and analyze your cells by FACS.
I agree suggestion from Pam Kimball and Alexander Misharin . You can use the high-content ArrayScanTM fluorescence microscopy. See reference from your research group, “Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScanTM assays “ , F. Gasparri et al. / European Journal of Cell Biology 88 (2009) 779–785
I can you extract/isolate the cell membrane first. Then from this membrane you can extract the proteins and then with this crude protein extract from the membrane you can run a western blot. All this might take little long time. Easiest and fastest way would be as suggested above, Fluorescence Microscopy.
Good luck
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