Journal: MethodsX

Article Title: A high throughput proliferation and cytotoxicity assay for co-cultured isogenic cell lines

doi: 10.1016/j.mex.2022.101927

Figure Lengend Snippet: Co-culture proliferation and cytotoxicity assay performance in high throughput 384-well format. An equal mixture of WT and PTEN KO cells (750-900 total cells/well) were plated into 384-well plates. All wells were treated with DMSO (0.01% final) to simulate compound screening, with columns 1 and 2 serving as DMSO controls for calculations and benzethonium added to columns 23 and 24 as a qualitative cytotoxicity control (not plotted). Data shown were based on the 72 hr time point after DMSO addition for columns 3-22. The relative KO cell count (●), an indicator of cell proliferation, was determined as the fraction of KO cells to total cells counted per well normalized to DMSO control wells. Differential KO cell cytotoxicity (■) was determined by calculating percentage of counted KO cells that were dead (green) minus the percentage of counted WT cells that were dead. Plate 2, row P data was not plotted due to a liquid handling automation error. The data from two 384-well plates were plotted that represent N=2 independent experiments performed on different days.

Article Snippet: Equipment • IncuCyte S3 Live Cell Analysis System (Sartorius, Ann Arbor, MI) • Water Jacket CO 2 Incubator (ThermoFisher) • Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA) • D300 Digital Dispenser with T8 Cassettes (Tecan, Morrisville, NC) • Centrifuge (Beckman Coulter) • Multidrop (ThermoFisher) • Pintool head and automated liquid handling (Biomek NX, Beckman Coulter) Reagents • CellTox Green (Promega, Madison, WI, cat # G8731) • Paclitaxel (Sigma-Aldrich, St. Louis, MO, cat# 33069-62-4) • MycoAlert kit (Lonza, Basel, Switzerland) • trypsin-EDTA (Corning, cat # 25-053-CI) • DMEM/F12 (Hyclone, cat # SH30023.01) • L-glutamine (Hyclone, cat # SH3003401) • HEPES (MP, # 1688449) • horse serum (Hyclone, cat # SH30074.03) • insulin (Gibco, cat # 12-585-014) • EGF (Sigma, cat # E9644) • Hydrocortisone (Stemcell cat # 07904) • cholera toxin (Sigma-Aldrich, cat # 9012-63-9) • penicillin/streptomycin solution (Gibco, cat # 15-140-122) • puromycin (Acros, cat # 58-58-2) • DMSO (Corning, cat # 25-950-CQC) • Benzethonium (MP, cat # 221661) • DPBS/MODIFIED (Hyclone, cat # SH30028.03) Software • IncuCyte Cell-by-Cell Analysis Software Module (Sartorius, Ann Arbor, MI) • Prism 9 software (Graphpad) Plasticware • 96-well black walled, clear bottom plates (Corning, cat. #3603) • 384-well black walled, clear bottom plates (Corning, cat. #3764) • Corning T150ml flasks (cat # 10-126-34) • 50 ml conical centrifuge tubes (Corning, cat # 4490) • 15 ml conical centrifuge tubes (Corning, cat # 430790) • 75 mL Flask (Corning, cat # 07-202-000) • Microcentrifuge Tubes, Eppendorf, 0.6 mL (Fisher Scientific, cat # 02-681-311) • Pipet Tips, Sterile, P1000 BIOTIXTM (Rainin, cat # 12-111-369) • Pipet Tips, Sterile, P200 BIOTIXTM (Rainin cat # 12-111-367) • Pipet Tips, Sterile, P20 BIOTIXTM (Rainin cat # 12-111-366) • Pipette, Aspirating, 2 mL (Corning, cat # 357558) • Pipette, Serological, 10 mL (Corning cat # 4488) • Pipette, Serological, 1 mL (Corning cat # 356521) • Pipette, Serological, 25 mL (Corning cat # 4489 • Pipette, Serological, 2 mL (Corning cat # 4486) • Pipette, Serological, 5 mL (Corning cat # 4487) • Reservoir, 50mL (Corning cat # 4871)

Techniques: Co-Culture Assay, Cytotoxicity Assay, High Throughput Screening Assay, Control, Cell Counting