Cyquant nf

Live/dead and CyQuant viability assays were performed as previously described (12 (link)). For live/dead assays, hBMECs were costained with calcein-AM (live/green, 3 μM; Invitrogen) and propidium iodide (dead/red, 2.5 μM; Calbiochem). calcein-AM-positive versus PI-positive cells were resolved using an Olympus IX51 microscope and overlaid using Adobe Photoshop. hBMECs were incubated with CyQuant-NF (Thermo Fisher), and fluorescence was quantified using a BioTek FLx800 fluorimeter.

Relative proliferation of cells was studied, using the CyQUANT®NF assay (Invitrogen, Carlsbad, CA, USA). Briefly, 500, 1000 and 3000 cells of HEC-1-A and HEC-1-A-S18-2 were grown in triplicates in a 96 well plate overnight in IMDM medium. After medium was removed, an aliquot of fluorescent dye was added and cells were incubated for 60 min at 37°C. After incubation, the fluorescence intensity was measured at λ=530 nm, using microplate reader Omega (BMG Labtech, Ortenberg, Germany). Percentage proliferation was calculated for HEC-1-A-S18-2 in comparison with control cells HEC-1-A, taken as 100%.

NIH3T3 and HT1080 cells were seeded in a 96-well plate at 1000 cells/well and incubated for 4 h. Growth medium was then carefully removed and 1× CyQUANT NF (Invitrogen) dye reagent was added. Cells were then incubated at 37°C for 1 h. This incubation period is required for equilibration of dye–DNA binding, resulting in a stable fluorescence endpoint. The fluorescence intensity of each sample was measured every 24 h using a fluorescence microplate reader with excitation at ∼485 nm and emission detection at ∼530 nm. Fresh growth medium was added every 48 h. Cell numbers at each time point were determined using a standard curve as per the manufacturer's instructions.

The materials used were L-NMMA 1 mМ (Sigma), 1-МТ (Sigma), antihuman ICAM-1 SC-107L (Santa Cruz), antihuman IDO (H-110 sc-25808, Santa Cruz), antihuman iNOS (aa 781-798, Clone 2D2-B2, MAB9502, R&D Systems), antihuman CD28 NA/LE (BD 555725), antihuman CD3 NA/LE (BD 555336), antihuman CD4 (558116, BD PharMingen), antihuman CD25 (555346, BD PharMingen), antihuman CD45 (304026, BioLegend), antihuman CD3 (300431, BioLegend), antihuman CD90 (328110, BioLegend), antihuman CD 73 (344006, BioLegend), antihuman HLA-DR (307610, BioLegend), transwell permeable membranes 0.4 μm (Greiner 665640), TRIzol® Reagent (Invitrogen, 15596-018), CyQUANT®NF (C35006, Invitrogen), Maxima SYBR Green/ROX qPCR Master Mix (2X) (K0221 Thermo Scientific), SuperScript® III Reverse Transcriptase (Thermo Scientific), and SuperSignal™ West Pico Chemiluminescent Substrate (34078 Thermo Scientific).
All donors were notified and agreed to take part in the research according to the draft Declaration on Bioethics and Human Rights (SHS/EST/05/CONF.204/3 REV) paragraph 6 items b and c. All operational procedures and protocols were strictly followed in accordance with MSU bioethics committee guidelines. All experimental protocols were conducted according to the GLP rules signed by the Ministry of Health (Order number 267 from 19 June 2003).