From: Holbeck, Judy (JHolbeck@ahs.llumc.edu)
Date: Fri Jan 31 2003 - 11:25:06 EST
Thank you for all the responses! The general consensus is that it's fine for a reagent control but it's not "inducing" apoptosis. One of the interesting things is that the cells only had 15-20% Annexin+/PI+ 5% ETOH treated and 6% Annexin+/PI+ untreated control cells. I expected to see the majority of the cells Annexin+/PI+ as many of the replies indicated. The PI+ staining is very clear cut. One thing I don't see is the morphological changes I expected, however, I read (Common Methods for Measuring Apoptotic Cell Death by Flow Cytometry, Purdue CD-rom Series, volume 3-special thanks for having this available on line!) not all cell models will exhibit the high SSC low FSC pattern. As for the cells being Annexin+, yes, they exhibit 60-65% Annexin+ 5% ETOH beyond unstained cells, HOWEVER, the cells are 50% Annexin+ UNTREATED control cells! The researcher is using an adherent cell line and using .01% Trypsin (still kind of curious why everything isn't Annexin+/PI+??? and yes, I gave them the search I did on the Purdue site pertaining to Annexin V and adherent cell lines). Both issues are part of an ongoing discussion about what they are trying to achieve, but I believe we've at least solved one part of my discussion with the researcher which is the 5% ETOH treatment is a staining/reagent control. Previously (but not in our lab), this researcher has had good results (i.e. high Annexin+%/low PI+%) with 5% ETOH in a nonadherent cell line. This being the 3rd attempt (2 different adherent cell lines and I gave them the website info before starting!), I have suggested they focus on trouble shooting or solving the background Annexin+ staining by finding an appropriate protocol, assay, or move to a nonadherent cell line (but sometimes I'm just here to move the cursor...one issue down ten to go.). Here is a summary of responses: As you probably are aware cell cycle protocols use ethanol to permeabilize cells. Not considering if these are trypsin released adherent cells, I would not recommend using ETOH with Annexin V. It will give positve results alright, but not because apoptosis has been initiated but rather, as you say, the membrane has been damaged. Interestingly this should be reflected in the fact that that one should see positive PI staining AND positive Annexin V staining which suggests necrosis. I wonder what your investigator sees? I would be interested to know. We recommend 3 apoptotic inducers with an assay. For example, Staurosporine, Camplothecin, and Cisplatin, or whatever is suitable for the cell type. I think your user is using this method (without realising it) as a means for generating a staining control.which is different form a biological control. This small etoh treatment probably alters the plamsa membrane integrity sufficiently to allow binding of annexin to the PS residues. For a biological control, for apoptosis induction, well there are severl methods depending on the target cell type but I would use etoposide (VP-16) or something simple like 1 mM h2o2 Some years back, an investigator here was looking at the effects of EtOH on various cell functions. I don't recall all the details, but apoptosis from various exposures to EtOH was one of the things he was measuring. I don't know what concentration or times that he used. Maybe your researcher has come upon a new inducer. Let us know what you find out. I've seen it listed in a catalog (Calbiochem, I think) as an inducer, but at that high a concentration I'd bet it is just oncosis. As a reagent control, it's fine, since it doesn't matter how the cells are killed. Do you see any Ann-V+/PI- with this treatment? The annexin V-FITC/PI is strictly a membrane integrity assay. In live cells the PI is excluded. Cells do not express phosphatidylserine on their cell surface, unless undergoing apoptosis. The normal residency for phosphatidylserine is in the inner cytoplasmic membrane. Treating with ethanol breeches the cytoplasmic membrane and exposes the phosphatidylserine for labeling and allows the PI to enter the cell. Treating with ethanol is a way to make a positive (necrotic) control that should be both positive for Annexin binding and PI uptake, all strictly inner-cytoplasmic. Hope this helps. I wouldn't use Etoh at all unless I wanted to fix my cells I would suggest low concentrations of hydrogen peroxide. There are references out there on this, and I have done it myself..... Ethanol just permeabilizes the cell, letting the annexin V bind to the phophatidyl serines on the inner side of the cell membrane. The cells will stain with both the annexin V and the PI, of course. Since the purpose of the kit is to detect apoptotic cells (annexin V+, PI-), not dead ones, this doesn't really serve as anything more than a staining control (gives you something nice to set compensation with, I suppose). To actually test their ability to enumerate cells undergoing apoptosis, they need to use one of the variety of pharcologic agents that induce programmed cell death. But as long as they have the correct controls in their experiment (i.e. untreated mice vs treated mice), they should be able to use the ethanol fixed cells for instrument setup, and the experimental samples should provide enough information to interpret their results. They may consider confirming their results with a second technique (fluorometric caspase 3, TUNEL, etc.) once they have some preliminary data. Thanks again, Judy Holbeck LLUMC, Immunology Center Flow Cytometry Tech, QCYM 909.558.8181 ext. 45407 Judy Holbeck LLUMC, Immunology Center Flow Cytometry Tech 909.558.8181 ext. 45376
This archive was generated by hypermail 2.1.6 : Thu Jan 01 2004 - 17:43:29 EST
