Hi C., Most, if not all, i.c. staining procedures call for a fixation step prior to permabilization. All wash steps in "standard" protocols are typically done with some agent included in the buffer that keeps the cells permeable; implying at least to me the membrane closes again. I also wouldn't worry about the leftover i.c. mAb and fluors affecting viability that much; it's the fixation that did them in well upstream that's the problem. If anyone knows of a protocol that managed to do i.c. and leave the cells viable, I'd be delighted to hear about it as well! Guy Guy Hermans Principle Scientist ________________________________ Ablynx nv Technologiepark 4 Tel +32 (0)9 262 00 00 9052 Zwijnaarde Fax +32 (0)9 262 00 02 Belgium Cell +32 (0)486 78 85 51 guy.hermans@ablynx.com www.ablynx.com ________________________________ -----Original Message----- From: Cinzia Mastrorilli [mailto:cmastror@vet.k-state.edu] Sent: Tuesday, June 10, 2008 17:56 To: cyto-inbox Subject: intracellular staining and culture Hi, I've a silly question: it is possible to culture cells that have been sorted by intracellular staining? Does the permeabilization process affect the functionality of the cells? I think they could be able to seal the membrane again, but what about the conjugated monAb that is present in the cytoplasm? Thank you CinziaReceived on Thu Jun 12 13:38:00 2008
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