Dear Flowers, I am posting the responses and valuable suggestions that I got for my questions regarding the CD107a/b expression. I am planning to try out the few combinations that have been suggested and will share the results as soon I solve the problem. Thanks for all your time. My special thanks to people who took time to respond to me personally. Best ******************************************************************** Vijay Nandakumar, Cellular and Molecular Immunology Laboratory Mayo Clinic 200, 1st street SW, Rochester, MN, 55905 Ph: 507-284-2128 (Direct) Lab Secretary: 507-284-4055 Pager: 127-10415 Cell: 865-406-6788 ********************************************************************* Are you using fresh or frozen cells? If they were frozen, try to add a dead cell exclusion marker. This really cleans up background on my CD107a. This is sold by invitrogen on a few colors. here is the order info to use it on the violet laser. If you have a FACS calibur and you can fit it into a panel it comes on other colors too. vividye 41178A INVITROGEN/MOL. PROBES Cox,Kara S, Merck Vijay, The high background staining, particularly to CD107, is coming from dead cells, which nonspecifically binds MAB's and is within your lymph gate. For this reason it is important to add a viability marker in all of your experiments. Recently, we switched to an amine reactive dye, which is much brighter and easier to use than EMA. See attached procedure. SP Stephen P. Perfetto, MS.,MT. (ASCP) Manager, Core Flow Cytometry Facility Vaccine Research Center, NIH Building 40 40 Convent Dr., Room 5507 Bethesda, MD 20892-3015 Perhaps your time of stimulation is too long ? Make shure also to add Monensine or Brefeldine during stimulation to avoide degranulation. You can also try a serum-free media to diminish the nonspecific activation Maybe that will help Good luck Michal Abel Hi Vijay, Were these fresh or frozen cells that were tested? We have run parallel ICS experiments that demonstrate less background for samples that are thawed and then rested in culture vials overnight vs. samples that are thawed and stained immediately. Also, is there a way to incorporate a viability dye into your experiment? Dead cells in the lymph gate will nonspecifically bind with CD107a. Once we added a viviability dye to gate out the dead cells, we saw much cleaner backgrounds in our unstimulated tubes. Laurie Lamoreaux Immunology Core Laboratory Vijay, I would need more info to be able to advise. Have you read our recent paper on this exact subject? If you want to send me your protocol and soem representative data I may be able to comment. Thanks, Simon. Vaccine Research Center, NIAID/NIH Bethesda, MD 20892 phone: (301)594-8633 llamorea@mail.nih.gov Hi, How long do you activate the cells? If it's short term then there shouldbn't be any dead cells? What is the conc. of Abs and what fluorophors are you using? Smita
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