another annexin question - (Sep/23/2006 )
I have been trying to analyse apoptosis of C2Bbe1 cells (clone of caco 2 cells) with flow cytometry using double labelling with annexin V and PI but i never seem to get any annexin positive cells - even in a heat treated cell control sample. I do get some double stained cells, but not any with just annexin. Initially I thought there might be a problem with the annexin but I tried using a different cell line and it worked just fine - is there any reason anyone can think of why annexin V is not binding the C2 cells? Has anyone had a similar problem? 
how ist annexin V labeled (FITC, EGFP...)?; check activity of your annexin V at adherent cells induced for apoptosis/necrosis with fluorescence microscopy; may be activity (binding and/or fluorescence) or concentration of annexin V is too low; apoptosis should be detectable
I need to perform the Annexin V staining to Adipocytes (differentiated from 3T3-L1). But i have a little worry with collecting the suspension cells. I wana collect cells by trypsinization. However, one paper recommended that "Trypsinization of adherent cells has been found to induce binding of annexin, leading to false-positive results. Instead, cells are detached by scraping after Annexin - PI incubation and apply flow cytometry with this suspension[i]"
Because fully differentiated adipocytes are so sensitive, I can't scrap them like this. And I'm also affaid that adipocytes will float in media eventhough I use any method.
Could you give me some advices?
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Because fully differentiated adipocytes are so sensitive, I can't scrap them like this. And I'm also affaid that adipocytes will float in media eventhough I use any method.
Could you give me some advices?
*^^*
what is the reason to detach cells? do you like to perform FACS analysis? as control perform analysis in 384/96-wells with adherent cells for fluorescence analysis
Yes, I wanna perform FACS.
By the way, if i want to see the cell - morphology after incubating with Annexin V and PI by using microcopy, what kind of 96 well - plate i can use?
What does 384/96 wells mean? (
Maybe, this is a silly question for you. But I never do experiment with fluorescence dyes, so that I confuse...) what is the reason to detach cells? do you like to perform FACS analysis? as control perform analysis in 384/96-wells with adherent cells for fluorescence analysis
Yes, I wanna perform FACS.
By the way, if i want to see the cell - morphology after incubating with Annexin V and PI by using microcopy, what kind of 96 well - plate i can use?
What does 384/96 wells mean? (
Maybe, this is a silly question for you. But I never do experiment with fluorescence dyes, so that I confuse...)it is a little bit advanced:
you need a plate reader for fluorecence (Perkin Elmer/Wallac or others)
as they only read 96-well or 384 well plates, you may try to culture your cells in special plates (suitable for fluorescent measurement and cell culture) with 96 or 384 wells; but then there is no need to detach cells; advanced plate fluorimeter may also run a spectrum of emssion
by the way, some succeed with detaching there cells by only using EDTA without trypsin;
BUT in any case of detachment there are effects on cell morphology and cell signalling as it is stress to the cells, and stress induces so much in a cells!
Thank for your advance, Kosmodrom.
I seeded the cells in 96 well -plate which is used for fluorescence from NUNC. It's a little difficult to control the experiment because i can't test the cell morphology during differentiation process by using normal microscope. 
I seeded the cells in 96 well -plate which is used for fluorescence from NUNC. It's a little difficult to control the experiment because i can't test the cell morphology during differentiation process by using normal microscope.
96 wells should be no problem with a light/phase contrast microscope; f.i. we dilute potentially stably transfected cells and seed them to 96-wells plates for microscopic inspection;