-------- On Tue 3/28/00 17:10 [cloxford@UCDAVIS.EDU] wrote -------- I was wondering whether anyone has used either Annexin V or TUNEL flow cytometry on VERY adherent epithelial cell lines in human or non-human species. . . . Without the aid of scraping and in the presence of only trypsin, it takes about 7 minutes to get these cells to lift off of a plate. I am concerned that this either form of cell harvesting will cause enough cell membrane damage to cause the assays to be inaccurate. That's exactly my problem. The majority of carcinomas I work with are extremely adherent. Even 0.05% Trypsin doesn't always help to dissociate all the clumps. Sometimes, I need the cells to be collected without use of enzymes and that's when the real fun begins. Short incubation time with 2 mM EDTA usually gives me an unacceptably low yield and long incubation often reduces viability below 50%. I found that supplementing EDTA-PBS with 10 mM Glucose and 25-50 mM HEPES can remarkably improve viability but it doesn't work all the time. Therefore, I spent some time searching Medline and found this: Menssen HD, Herlyn M, Rodeck U, Koprowski H. "Rapid dissociation of adherent human tumor cells by ultrasound". J Immunol Methods 1987 Nov 23; 104(1-2):1-6. PMID:3316390. Here's a quote. "For ultrasonic detachment, tissue culture flasks containing washed cells in 100 ul of modified HBSS / cm(2) were sealed in Parafilm and immersed approximately 0.5 cm into a water-filled ultrasonic cleaner (Astron model 9, 100 W output), operating at 43 kHz, and sonicated at RT for 10-50 sec. Brief tapping against the flask after half of the sonication time loosened the cells from the substrate." Recipe of modified HBSS: Ca2+/Mg2+ free Hanks' BSS + 30 mM HEPES + 0(!) mM EDTA. Did anybody try that method? May I ask the folks with ultrasonic cleaners to email me with their experience? Thanks for helping, Yuri Kudinov, postdoctoral fellow SVMC, Immunotherapy Lab 201 S.Alvarado St., Ste 312 Los Angeles, CA 90057-2354 ------------------------------------------ Menssen HD, Herlyn M, Rodeck U, Koprowski H Rapid dissociation of adherent human tumor cells by ultrasound J Immunol Methods 1987 Nov 23;104(1-2):1-6. PMID:3316390 Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104. ABSTRACT. Cultured human melanoma and gastrointestinal carcinoma cells were detached from substrate and further dissociated by placing the culture vessel into a water-filled ultrasonic cleaner (43 kHz) and sonicating it for 10-50 sec. Plating efficiency and long-term growth of three melanoma cell lines were similar after ultrasound or trypsin detachment. Binding of monoclonal antibodies that define normal and tumor-associated antigens on melanoma and colorectal carcinoma cells was not affected by ultrasound in 21 out of 23 cases. The 40 kDa colorectal carcinoma-associated antigen defined by monoclonal antibody CO 17-1A was more highly expressed after ultrasonication than trypsinization. The antigen defined by antibody CO 44.1 on these cells was more sensitive to sonication. This method represents a rapid, effective and gentle alternative to trypsin detachment of cultured cells, especially when repeated cell washing or centrifugation steps are required. -----Original Message----- From: Carol Oxford [mailto:cloxford@UCDAVIS.EDU] Sent: Tuesday, March 28, 2000 17:10 To: cyto-inbox Subject: Annexin V and TUNEL I was wondering whether anyone has used either Annexin V or TUNEL flow cytometry on VERY adherent epithelial cell lines in human or non-human species. Specifically, I am looking for a reliable method for detecting apoptosis in normal mammary gland epithelial cells from domestic cats. Without the aid of scraping and in the presence of only trypsin, it takes about 7 minutes to get these cells to lift off of a plate. I am concerned that this either form of cell harvesting will cause enough cell membrane damage to cause the assays to be inaccurate. I would appreciated any feedback, and suggestion of a manufacturer and/or a protocol would be extremely helpful. Thanks, D. McAloose
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