Hey ho Flow-ers, We have an established phagocytosis protocol, which HAS worked well for us. The organism being used is Mycobacterium avium-FITC labelled (MA-FITC), which is phagocytosed by HIV- and HIV+ monocytes. (a commercial quenching agent is being used.) Of recent times we have switched to a Becton Dickinson (BD) CD14-PE antobody (Ab) from a DAKO CD14-PE. The problem encountered is that where as once I used to get staining at 37C with DAKO CD14, the BD Ab fails to do this at the same temperature, however the BD Ab stains beautifully at 4C. So the dilemma? Has anyone come across inhibition of binding of BD Abs, (or any other Ab for that matter) when working with MA at 37C (or any temp). Is it common for BD Ab to become inactive if kept at 37 for only 10mins (in our protocol) with MA or any other organism?? Does MA have properties which inactivate this particular BD CD14-PE...at 37C?? Is there a chance of MA digesting PE at 37C, rendering it non fluorescent but still bound?? (and oh yes, the antibody hasn't expired yet) Fanx... In advance Geza Paukovics - Flow Laboratory - AIDS Pathogenesis Research Unit Macfarlane Burnet Centre .***. .***. .** PO Box 254 _--_|\ * | | | * * | | Fairfield, VIC 3078 / \ * * | | | * * | | | AUSTRALIA. \_.--._/ * * | | | * | | | * ph. (+61 3) 9282 2132, O '***' '***' FAX (+61 3) 9282 2100
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