Celltiter glo system

Human lines were maintained in RPMI media (Gibco) supplemented with 10% FBS, penicillin and streptomycin. All murine lines were derived from fetal liver cells infected with MLL-ENL and NRas^G12D or Flt3 ITD expressing vectors [20] (link). Murine lines were maintained in stem cell media (40% IMDM, 40% DMEM, 20% FBS, with or without murine SCF 10 ng/mL, murine IL-6 2 ng/mL, and murine IL-3 0.4 ng/mL). Viability assays were carried out according to the manufacturer's protocols with the Cell Titer-Glo system (Promega).

For the NFAT reporter gene assay, GloResponse™ NFAT-RE-luc2P HEK293 cells (Promega) were seeded at a density of 40,000 cells per well in FreeStyle medium (Gibco). DutaFabs were pre-incubated with VEGF (20 ng/mL), incubated for 30 min and added to the cell suspension. After 5 h incubation at 37 °C, BioGlo reagent (Promega) was added according to the manufacturer’s instruction and luminescence was read using an Infinite Pro microplate reader (Tecan). For the HUVEC proliferation assay, human umbilical vein/vascular endothelial cells (HUVEC) (ATCC) were seeded on collagen-coated 96-well plates (ACEA Biosciences) at a density of 3000 cells per well and incubated in starvation medium (Promocell) for 24 h. DutaFabs were pre-incubated with VEGFA-165 (8 ng/mL), incubated for 30 min and added to the cell suspension. Proliferation was quantified after 48 h with the CellTiter-Glo® system (Promega) according to the manufacturer’s instructions using an Infinite Pro microplate reader (Tecan).

(i) FVS780 LIVE/DEAD assay. The BD Horizon fixable viability stain 780 (FVS780) assay (BD Biosciences) was used to determine plasma membrane permeability. Culture supernatants were preserved for analysis of detached cells. Cells were then washed with PBS and detached with TrypLE Express. Harvested cells were washed with PBS and stained with BD Horizon FVS 780 as per the manufacturer’s protocol. Viable and dead cells were then analyzed and quantified using flow cytometry.
(ii) CellTiter-Glo assay. The CellTiter-Glo system (Promega) was used as per the manufacturer’s protocol to determine the number of viable cells based on their metabolic activity. At the indicated time points postinfection, CellTiter-Glo reagent was added to each well in equal volumes. Contents were mixed for 2 min to induce cell lysis, followed by a 10-min incubation to stabilize the luminescence signal. The resultant luminescence was measured using the Synergy H1 microplate reader in 96-well solid white plates with an integration time of 1 s per well.

The cells were plated into a 96-well plate (1 × 10⁵ cells/well in 100 μL of medium) and were cultured for 18 h. Cell viability was monitored using the luminescent-based CellTiter-Glo system (Promega Corporation, Madison, WI, USA) according to the manufacturer's recommended protocols [16 (link)]. The luminescence of each well was read using an Infinite M200 multimode microplate reader (TECAN, Switzerland). Cell viability was normalized and expressed as a percentage of the untreated cells [17 (link)].