Open AccessTime‐related effects of enzymatic disaggregation on model human lung carcinomas
Author(s) -
Howard Randy B.,
Shroyer Kenneth R.,
Marcell Tere,
Swanson Lucille E.,
Pagura Marco E.,
Mulvin David W.,
Cowen Michael E.,
Johnston Michael R.
Publication year - 1995
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990190209
Subject(s) - collagenase , trypan blue , cytology , microbiology and biotechnology , flow cytometry , hyaluronidase , enzyme , andrology , peripheral blood mononuclear cell , chemistry , biology , cell , pathology , in vitro , biochemistry , medicine
Enzyme cocktails used to prepare tumor cell suspensions may influence yield, viability, and cytology, thus time‐related cocktail effects on model human lung carcinomas were examined. A549, NCI‐H125, and NCI‐H460 carcinomas were completely disaggregated at 25°C over 2 h with either (mg/ml) collagenase/DNAse (C/D, 1/0.1), collagenase/hyaluronidase/DNAse (C/H/D, 1/0.1/0.1), or polymyxa protease/DNAse (PP/D, 3/0.1). Trypan blue viabilities, total yields, viable yields, and flow cytometric percent tumor cells (TC) were measured every 20–30 min (n = 4–7 per tumor type). The final percentages of TC, mononuclear cells (MN), polymorphonuclear cells (PMN), lymphocytes, and necrotic cells were determined by cytology (n = 4–5 per tumor type). The time‐dependent measurements showed that (1) disaggregation was progressive and complete with all cocktails; (2) viability was stable or increasing with all cocktails; (3) percent TC was stable for all cocktails, but lower for PP/D than C/D in final suspensions; and (4) PP/D gave lower final total yields, higher final viabilities, but the same final viable yields as the C cocktails, suggesting selective elimination of dead cells by PP/D. Final cytology measurements showed that PP/D gave a lower percent MN and a higher percent PMN than C cocktails. Cocktail effects may importantly influence cell suspension properties. © 1995 Wiley‐Liss, Inc.