Hi, We do cell cycle analysis on fixed nonadherent cells, about 75,000 cells per well of a 96-well plate suspended in about 120 microliters of buffer when it goes onto the Cytek AMS autoloader. We treat with RNase, stain with propidium iodide, and look at FL2-A histograms to measure the proportion of cells in G1, S, and G2/M. With this protocol, the Cytek autoloader takes about 45 minutes to go through 96 wells (6000 events per well--but going to 10,000 events would only increase the time by a few seconds per well, because most of the time is required for washing the probe and moving the plate around, not taking the sample). We've never tried this with adherent cells in a 96-well plate, but my guess is that you'd have a hard time getting enough cells to analyze 10,000 events per well. Maybe a 24-well would give you enough starting material to move into a 96-well after you've released the cells from the plate. I'd guess that about 30,000 cells per well would be enough, and maybe less. The 96-well loader fits under the FACSCalibur. You remove the panel behind the sample input tube and most of the loader fits in the large open space underneath the FACSCalibur. The front part of the loader can slide back behind the sample input tube so you can run single tubes if necessary. When you want to run 96-well plates, you slide the loader forward a little bit and attach a short piece of tubing to the sample input tube, which takes about 30 seconds. There's a control box (1-foot cube) that sits just to the right of the FACSCalibur and 2x 1 liter bottles for wash and waste liquids that fit behind. I believe this unit goes where a carousel loader would go, but with a longer piece of tubing and longer sample boost times it could probably go onto the bench next to the cytometer. Better check with Cytek (cytekdev@aol.com) on this one. Laird Bloom Phylos, Inc. 128 Spring St. Lexington, MA 02421 > ---------- > From: Ellen.Freed@astrazeneca.com > Sent: Thursday, October 11, 2001 4:22 PM > To: Cytometry Mailing List > Subject: RE: 96 well FACS - for Cell Cycle analysis > > > HI All > > A number of you have responded to my question about this a week or two > ago. > . .Thanks. But it seems all this might be impossible if you're doing cell > cycle analysis. Has anyone actually grown enough adherent cells in 96 > well > format in order to trypsinize and fix and stain all in the plate and do > your > analysis from that same plate? - Assuming you want to analyze at least > 10,000 events - after gating out other stuff. I've always started with a > lot more cells than I grow in 96 plate wells. > > Also, even if you grow in other plates and transfer, the cells would have > to > be at high concentrations and run slowly - so it would take a long time. > Somebody mentioned running a plate in 45 minutes - this is for what sort > of > analysis? > > Also, is the 96 well attachment easily interchangable with a carousel or > just a single tube loader??? > > Thanks much. > > Ellen Freed > > Ellen Freed > Scientist > AstraZeneca R&D Boston >
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:57:57 EST