Dear fellow Flow-ers, A while back I asked for advice about how to prevent cell clumping together during an apoptosis (propidium Iodide) assay. I've put together a summary of the advice given in the hope that it may be of value to others in the future. We tried altering our procedure and while we were clump free we were also cell free but it is early days yet. Thanks for your help. Regards, Jeff Barry Paterson Institute, Manchester, UK General tips: Firstly, if they are adherent or even if they grow in suspension but in clumps, make sure that they are in as good a single cell suspension that you can make before fixation A biodegradable anticoagulant named ACD-A at 0.6% in PBS prevents cell clumping of mononuclear blood cells and some stromal cells. Also it can be a good idea to filter the cells through a 60-70um mesh to get rid of the larger lumps (although clearly this will lose cells). Keep cells suspended in 1 to 5 % BSA/media helps prevent clumping. Try adding some EDTA (0.02%) Try adding DNase at 50U/mL in all preparation media. If you are using an alcohol fixation, try to resuspend the cells in the last remaining volume after centrifugation and then add the alcohol drop wise with mixing. Another problem we have run into is over trypsinization. It sounds as if you are getting free DNA into your prep which will string everything together. We have experienced this also. Final tip may be to assure yourself that the cell number is kept below 1X10e6/ml. The cells tend to clump less for some reason. Where visible clumps are seen in the sample tubes, our users use nylon mesh or Falcon 2270 or 2235 tubes with the mesh in the caps to filter the samples. If the cell type they are using tends to clump, Accutase sold by Phoenix Flow Systems o San Diego, CA. USA prevents clumping. They also sell Accumax, an enzyme based product used for creating single cell suspensions from tissue samples. Ethanol fixation step: Inadequate ethanol fixation is a primary cause of clumpage - it's a good idea to vortex while fixing (careful though if looking for apoptotic cells as they could explode at this point!). Also make sure that the pellet is as free as possible of residual PBS before adding the ethanol. Use EDTA or Dnase: Secondly you could try using either EDTA or a low level concentration of DNase in your PI solution as it is DNA from blown-up cells that can cause clumps as well. Use Anti-clumping agents: I have read about a firm that's called TCS CellWorks in the UK, that sells Accumax. It is used to disaggregate clumpy cells and viability seems to be very good after treatment. You can read about it on WWW.innovativecelltech.com <http://WWW.innovativecelltech.com> for additional information. You can also contact the office manager at TCS CellWorks Ltd :Fay Crook : Fay@tcsgroup.co.uk <mailto:Fay@tcsgroup.co.uk> PAA sell a product called Accutase, that's for prevent cell clumping. I never used it, so I can't tell you if this product works or not Use Nuclei: A variation on this assay utilizes hypotonic lysis to isolate individual nuclei instead of analyzing whole cells. It is described in: J Immunol Meth, 139: 271-279, 1991. This should eliminate cell/cell binding. Protocols: Protocol 1: APOPTOSIS AND CELL CYCLE ANALYSIS: Courtesy of Nigel Miller 1. 1 million cells in 200ul PBS 2. Add with stirring 2ml ice cold 70% ethanol 30% PBS. 3. Leave 30 mins. On ice. 4. Centrifuge 2000 rpm 5 mins. 5. Resuspend pellet in 800ul PBS. If cells are clumped pass through a 25 gauge needle. 6. Add 100 ul Rnase (1mg/ml-boiled 10' to destroy DNAse). 7. Add 80 ul Propidium Iodide (0.5mg/ml). 8. Incubate at 37 deg C for 30 mins. 9. Analyse using the FACSCalibr.. 10. Use MODFIT to calculate DNA. Protocol 2: Courtesy of Dennis Young Fix cells by adding the 10^6 cells (or less) in 1 ml to 10 ml ice cold 70% EtOH while vortexing. Use detergents (0.1% Triton-X 100) Use filters (20 - 75 microns should work) Use 25 gauge needle to resuspend cells right before sampling (Lastly) Lyse cells and measure nuclei instead. Protocol/Advice 3: Courtesy of Kent Claypool If preparing a single cell suspension is producing aggregates remove the cytoplasm through a pepsin digest (references below) and look at bare nuclei. Also the PI for a sub-G1 peak does not always work. The example at the below web site had cell death that didn't produce a sub-G1 peak until a positive control with Fas ligand was used. Then produced a great profile. Cells have to have Fas receptor to trigger this apoptotic pathway. Might want to also look at gel electrophoresis for DNA laddering, Bcl-2 by western and/or flow, as well as the 'TUNEL' assay which can be done with nuclei also. http://sciencepark.mdanderson.org/flow/files/DNA_PI.html <http://sciencepark.mdanderson.org/flow/files/DNA_PI.html>
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