Date: Wed Jun 28 2006 - 03:08:17 EDT
Hi Valentina, Miltenyi has MACS kits for both positive and negative one-step selection; don't bother with improvising your own two-step protocol as it will only cost more time and money on reagents. As for the procedure: just do as Miltenyi recommends regarding buffers, staining protocol etc. In my experience, you have little opportunity to improve on theirs (after all, they've been doing this for a long time) and at least it's optimized for their technology. Regarding the BSA, you'll have to think hard about the kinds of antigen your cells are "allowed" to have seen before and during culture - this depends on your experiment. If BSA is a no-go, try using foetal calf or inactivated human (autologous to the cells donor won't show cells many new antigens!) serum instead. Best of luck, Guy Next generation therapeutic <http://www.ablynx.com/> antibodies Guy Hermans, PhD Senior Scientist Ablynx NV Technologiepark 4 B-9052 Zwijnaarde Belgium guy.hermans@ablynx.com tel: fax: mobile: +32 (0)9 261 06 57 +32 (0)9 261 06 27 +32 (0)486 788 551 <https://www.plaxo.com/add_me?u=30065269879&v0=994426&k0=2009290972> Add me to your address book... <http://www.plaxo.com/signature> Want a signature like this? -----Original Message----- From: Valentina dal secco [mailto:valentina.dalsecco@uniroma1.it] Sent: Monday, June 26, 2006 4:03 PM To: cyto-inbox Subject: negatively selecting CD8 + T cells Dear all, I have to obtain pure CD8 for at least 5-days-long in vitro culture. Since I have the Miltenyi biotech "Pan T cell isolation kit" to get pure total T cells, after having obtained a T cell preparation, I can negatively select CD8+ T cells by labelling total T cells in sequence with anti-CD4-PE and Miltenyi biotech "anti-PE Microbeads" and therefore load them on MS columns to collect effluent. Do you think is this the method of election to get pure CD8 to culture (since I have no FACS sorter)? Do you have any specific suggestion regarding: - the maximum number of T cells to label with anti-CD4 and in which volume (to avoid sedimentation), - the antibody concentration (the same as for cytometry analysis labelling) - the labelling buffer (PBS-BSA 1% as for cytometry analysis labelling?) - the BSA should be 99% pure since I must culture CD8+ T cells afterwards? - should I keep MS colums cool before loading? - and finally, should I pass the selected fraction over a second freshly prepared column to increase purity? Thank you very much in advance Yours sincerely, Valentina Dal Secco Univ. of Rome ----------------------------------------------------------------------- THIS E-MAIL MESSAGE IS INTENDED ONLY FOR THE USE OF THE INDIVIDUAL OR ENTITY TO WHICH IT IS ADDRESSED AND MAY CONTAIN INFORMATION THAT IS PRIVILEGED, CONFIDENTIAL AND EXEMPT FROM DISCLOSURE. If the reader of this E-mail message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please notify us immediately at ablynx@ablynx.com. Thank you for your co-operation. "NANOBODY" and "NANOCLONE" are registered trademarks of Ablynx N.V. -----------------------------------------------------------------------
