caa a22 4500 445292997 CHVBK 20180317142534.0 cr unu---uuuuu 170323e20100201xx s 000 0 eng 10.1007/s11626-009-9256-8 doi (NATIONALLICENCE)springer-10.1007/s11626-009-9256-8 A protocol to effectively create single cell suspensions of adherent cells for multiparameter high-throughput flow cytometry [Elektronische Daten] [Evan Glazer, Katheryn Massey, Steven Curley] High-throughput flow cytometry of adherent cells is difficult because the creation of single cell suspensions can damage cells and yield artificial results. We describe a protocol to increase the single cell suspension yield of adherent human cells without injury. Doxorubicin, a cytotoxic agent, was administered to adherent human pancreatic carcinoma cell lines (Panc-1 and AsPC-1) to produce alterations in the cell cycle and intracellular protein expression. The cells in 96-well plates were disassociated using a collagenase and trypsin mixture. Fluorescence-activated high-throughput flow cytometry evaluated cellular viability as well as surface and intracellular protein expression. Cell cycle analysis was performed using 7-aminoactinomycin D and intracellular protein characterization was performed using a fluorescein-labeled monoclonal antibody against activated caspase-3. The collagenase-trypsin-based protocol increased single cell events from 31.9 ± 0.5% using trypsin alone (standard) to a range of 62.1% to 85.5% without adversely affecting viability. High-throughput flow cytometry demonstrated that the addition of collagenase to the disassociation solution not only permitted significantly higher rates of single cell creation, but it did not negatively affect the doxorubicin-induced protein expression. This protocol allows for expedient and effective disassociation of adherent human cells in order to investigate alterations in specific cellular enzymes and pathways. The Society for In Vitro Biology, 2009 High-throughput flow cytometry nationallicence Multiparameter flow cytometry nationallicence FACS nationallicence Collagenase nationallicence Glazer Evan Department of Surgical Oncology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 107, 77030, Houston, TX, USA aut Massey Katheryn Department of Surgical Oncology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 107, 77030, Houston, TX, USA aut Curley Steven Department of Surgical Oncology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 107, 77030, Houston, TX, USA aut In Vitro Cellular & Developmental Biology - Animal Springer-Verlag 46/2(2010-02-01), 97-101 1071-2690 46:2<97 2010 46 11626 https://doi.org/10.1007/s11626-009-9256-8 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11626-009-9256-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Glazer Evan Department of Surgical Oncology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 107, 77030, Houston, TX, USA aut NATIONALLICENCE 700 1- Massey Katheryn Department of Surgical Oncology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 107, 77030, Houston, TX, USA aut NATIONALLICENCE 700 1- Curley Steven Department of Surgical Oncology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 107, 77030, Houston, TX, USA aut NATIONALLICENCE 773 0- In Vitro Cellular & Developmental Biology - Animal Springer-Verlag 46/2(2010-02-01), 97-101 1071-2690 46:2<97 2010 46 11626 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer