Use for discussion of various microscopy and imaging techniques, such as phase, fluorescence and confocal microscopy, electron microscopy, and various other imaging techniques.
I am trying to set up a phagocytosis assay on Neutrophils using beads. I came with the conclusion I would need to try both flow cytometry and immunoflorescence. Is there a particular type of beads that can be used for both applications? instead of ordering two different types of beads?
I am currently trying it out with 3 micron latex beads and trying to observe phagocytosis under light microscopy.... I have problems with the opsonization, I am using autologous serum for the time being, but going to get some IgG form a clinical lab.
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- Joined: Mar 05 2011 8:40 am
I don't know about opsonization but i hope this is helpful:
I have used Fluorescbrite beads in cytometry. They give a bright signal and are relativeley stable (both inside cells and for several years in your fridge).
If you can, a quick way of doing this is:
-put beads on cells and incubate for several timepoints to make sure that cells take up the beads (for 1µm-diamter bead it starts as soon as 5-10 minutes).
- wash cells several times to take out excess beads and ones stuck on membrane (cells need to by resuspended if they are adherent);
- take one part of your suspension to flow cytometrer (dont forget the non-treated cells) or fix them in PFA;
-re-plate the rest for some time (at least one hour) in normal medium before fixation followed by microscopy.
Fluorescent beads are better and easier to handle for microscopy because you can co-stain your cells with a membrane dye (or with phalloidin if it's present in the cell cortex) to verify that the beads are inside the cells and not stuck on the outer side of the membrane. You can also do timelapse microscopy with this setup with realtime Z-stacks if you have a quick motorized apparatus on your micriscope.
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- Joined: Apr 15 2009 9:42 am
- Location: Lyon, France
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