Sorry, looks like I was a bit vague in my first email! We've been using unlabelled primary antibodies to CD14 with an RPE labelled secondary. The cells have been cultured overnight in phenol red free RPMI 1640 with 0.2% BSA (no FCS). I've been blocking Fc receptors using 10% human AB serum and blocking non-specific by using 5% BSA and 10% donkey serum (secondary is raised in donkey). At earlier passage numbers (up to 19) I had the PMT voltages at around 600V but now I am having to decrease them to around 450V. I tried running unstained cells (no IgG, no antibodies) and they too run about the same voltage. Should I be concerned that the PMT voltages need to be dropped so low? Thanks, Elaine Kennedy -----Original Message----- From: Kennedy, E. (Elaine) [mailto:E.Kennedy@organon.nhe.akzonobel.nl] Sent: Wednesday, May 15, 2002 12:24 AM To: cyto-inbox Subject: cell lines in flow cyt Hi all, I was wondering if anyone had any advice re. the use of cell lines in flow cytometry. I've been using monocytic cell lines (THP-1 and Mono Mac 6) and my protocol seemed to be working well however it seems to have went a bit askew of late. The cells are at passage numbers 21 and 25 respectively but otherwise appear to be healthy. Any advice much appreciated. Thanks, Elaine Kennedy Scotland -------------------------------------------------------------------- This message, including attached files, may contain confidential information and is intended only for the use by the individual and/or the entity to which it is addressed. Any unauthorized use, dissemination of, or copying of the information contained herein is not allowed and may lead to irreparable harm and damage for which you may be held liable. If you receive this message in error or if it is intended for someone else please notify the sender by returning this e-mail immediately and delete the message. -------------------------------------------------------------------- -------------------------------------------------------------------- This message, including attached files, may contain confidential information and is intended only for the use by the individual and/or the entity to which it is addressed. Any unauthorized use, dissemination of, or copying of the information contained herein is not allowed and may lead to irreparable harm and damage for which you may be held liable. If you receive this message in error or if it is intended for someone else please notify the sender by returning this e-mail immediately and delete the message. --------------------------------------------------------------------
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