(123) Assay Card

General Information
Summary Nonclonogenic fluorometric microculture cytotoxicity assay was performed on psyle A, C and E using U-937 GTB human lymphoma tumor cell line.
Condition The cell integrity was measured based on the fluorescence generated from hydrolysis of fluorescein diacetate to fluorescein by cells with intact membranes. The psyle peptides were dissolved in 10% EtOH, and 20 μL/well was dispensed onto 96-well microtiter plates (10 concentrations ranging from 30 to 0.5 μM) in triplicate with the experiment repeated in triplicate. Phosphate buffer solution (PBS) and Triton were included as negative and positive controls, respectively. To each well, 180 ?L of media (RPMI-1640, 10% heat-inactivated fetal bovine serum, 60 μg/mL penicillin, 50 μg/mL streptomycin, and 2 mM glutamine) containing U-937 GTB human lymphoma tumor cells (2 x 104 cell/well) was added, and microtiter plates were incubated (37 C, 5% CO2) for 72 h. Following Q2-buffer preparation (40 mL of 125-NaCl, 10 mL of 25 mM Hepes, 400 mL of Millipore H2O, 4 M NaOH), plates were centrifuged at 1000 rpm for 5 min and cells washed with PBS. After fluorescein diacetate (FDA) was added to preheated Q2- buffer (1 μL/mL), 100 μL of FDA/Q2-buffer was added to each well using the Multidrop 384 Labsytem followed by plate incubation (37 C, 5% CO2) for 40 min. Fluorescence was measured on a Labsystems Flouroskan II at 538 nm, following excitation at 485 nm, and inhibition of growth was quantified as a survival index (average fluorescence of test wells relative to control wells minus the blank) expressed as percent.
Result The IC50 values for psyles A, C, and E were 26, 3.50, and 0.76 μM, respectively.
AssayType Cancer Cell Toxicity

References
Gerlach SL, Burman R, Bohlin L, Mondal D, Göransson U (2010) Isolation, characterization, and bioactivity of cyclotides from the Micronesian plant Psychotria leptothyrsa J Nat Prod 73:1207-1213

Cross-references
Proteins Assayed psyle E
psyle A
psyle C