Department of Physics, Chemistry and Biology (IFM)

Methods

• Cytotoxicity test by MTS assay

MTS-assay is a colorimetric method to determine the proliferation of cells in culture. MTS is a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbomethoxyphenyl)-2- (4-sulphoneyl)-2H-tetrazolium, which is light sensitive and is bioreduced by cells into a formazan product that is water soluble. The amount of formazan product produced is relative to the number of viable cells in the well and can be measured as optical density at 490 nm.

Human Stromal cells of Corneal Limbus (hSCLs) were isolated from human donor corneas. The cells were grown in  75 cm² tissue culture flasks (Falcon, Becton Dickinson), with complete Dulbecco’s Modified Eagles Medium (DMEM) ( DMEM, 10% fetal calf serum (FCS) (PAA laboratories, Germany) , 1% penicillin (10,000 U/ml) (PEST)  and streptomycin (10000 µg /ml) (Thermo Scientific). They were maintained in a humidified incubator at 37ºC with 5% CO₂until the cells were confluent.

Prior to use, the passage 4 (P4) hSCLs were trypsinized using 0.05% trypsin-EDTA (GIBCO, Invitrogen) at 37ºC for 2-3 minutes. The cells were then centrifuged (Eppendorf Centrifuge 5804, Hamburg) at 300x g for 5 minutes. The pellet was suspended in 1ml of complete medium and then counted using an automated cell counter (Scepter^TMHanheld Automated Cell counter, Millipore). The suspended cells were seeded in a 96well plate at a density of 5000 cells/well by using a multichannel pipette. The cells were then incubated at 37ºC and 5% CO_2.for 6 hours. The anti viral compounds (Vironova AB) were dissolved in 10% Dimethyl Sulfoxide (DMSO) in dH2O which was heated to 85ºC and prepared at varying concentration depending on their solubility.

Posteriorly, varying concentrations of anti-viral compounds were added to the wells of the plate.  Each sample was done in triplicates along with 50% DMSO (Dead cell control), 0.1% DMSO (DMSO control) and complete medium without anti-viral compounds (Live cell control). The plate was incubated overnight (18 hrs) at 37ºC. After incubation, the plate was washed once with complete DMEM followed by addition of MTS according to manufacturer’s instruction (Promega) and incubated at 37ºC and 5% CO₂ for 2 hrs. The color change from yellow to brown due to the conversion of MTS into soluble formazan product was observed. Then, t he optical density was measured at 490nm directly in the 96 well plates using Victor³VMultilabelPlate Reader (Perkin Elmer Multilabel Counter, Wallac-1429-instrument). The quantity of formazan product is directly proportional to the number of living cells in the culture.  

• Direct Plaque Assay

Plaque assay is used to measure the ability of the virus to infect cultured cells and to determine viral titers. This technique is used to determine the ability of a single virus to form a “plaque” on a confluent monolayer of cultured cells. The culture is stained and the numbers of plaques are counted.

Green Monkey Kidney cells (GMK) was kindly provided by department of virology Sahlgrenska University Hospital (Goteborg). The cells were grown in a 75 cm² tissue culture flask (Falcon, Becton Dickinson), with complete medium. They were maintained in a humidified incubator at 37ºC with 5% CO₂ until the cells were confluent. GMK cells are prone to viral infection ( Vahlne & Lycke, 1978 ) and it has fibroblastic nature like hSCL cells.

The GMK cells were trypsinized and seeded on 24 well plates and maintained in a humidified incubator at 37ºC with 5% CO₂ until the cells were confluent. The cells were infected with herpes simplex virus type 1 (HSV-1) clinical isolate at 50PFU (Plaque Forming Unit)/100uL per well. The cells were infected for 1 hour at 37ºC with 5% CO₂ . After 1 hour of infection, the medium was removed and the plates were washed once with complete medium. Then 495uL of complete medium with 0.5% gamma globulin was added to each well. 5.0 ul of varying concentrations of antiviral compounds or appropriate controls were added to the wells according to Table 1. Each sample was run in triplicates and controls were run in duplicates (positive control without compounds, 0.1% DMSO (DMSO control) an uninfected control). The plates were incubated for three days at 37ºC. The medium was discarded and the plates were stained with Crystal Violet (crystal violet 1g (Acros Organics), Formalin (37%) 10 ml (Sigma Life science), Concentrated Acetic Acid 5ml (Merk), Ethanol (70%) 100 ml (Solveco)). The plates were rinsed with tap water and dried, after which t he numbers of plaques were counted by using regular microscopy. Antiviral effect was defined as being able to inhibit the formation of 50 % of the plaques as compared with positive control.

• Statistical Analysis

The cytotoxicity and antiviral activity of the B-220 compounds were analysed statistically through one way Analysis of Variance followed by Dunnet’s Test at p≤0.05 significance level.