Infinite m200 microtiter plate reader

The cell invasion was measured using the same Cytoselect 24-well cell invasion assay kit (Catalogue number CBA-100-C, Cell Biolabs, Inc., San Diego, CA, USA) with the variation of coating the upper surface of the insert membrane with a protein matrix isolated from Engelbreth–Holm–Swarm tumor cells. Then, the basement membranes of Boyden chambers were rehydrated with 300 μL serum-free media, and 1 × 10⁶ cells were then seeded into the upper area of the chamber in serum-free media (control) with or without the plant extracts. Overnight serum starvation had been performed prior to running the assay. Then, 500 μL of media containing 10% FBS was added to the lower well of the migration plate. After incubation for 48 h, the non-invading cells on the upper surface of the inserts were removed with a cotton swab and invading cells on the lower surface were stained with crystal violet Cell Stain Solution and incubated for 10 min at room temperature. Then, the inserts were gently washed in a beaker of water and left to air dry. Each inserts was transferred into wells containing 200 μL of Extraction Solution (10% acetic acid) and incubated for 10 min at room temperature on an orbital shaker. Then, 100 μL of each sample was transferred to a 96-well plate and measured at 560 nm by using an Infinite^® M200microtiter plate reader (Tecan).

Differential HERV expression and its repression by antiviral drugs were monitored using an indirect ELISA method. In brief, 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with protein homogenates (5 μg/100 μl) overnight at 4 °C. Well contents were aspirated and the wells washed 3 times with washing buffer (PBS/0.05 % Tween 20). The wells were then incubated with 300 μl blocking buffer [PBS/0.05 % Tween 20/1 % bovine serum albumin (BSA)] each at 37 °C for 1 h and then washed 3 times. Primary antibodies diluted 100 μl in blocking buffer 1:500 were added, followed by incubation at 37 °C for 1 h. The wells were aspirated and washed three times followed by incubation with an HRP-conjugated secondary antibody (Sigma-Aldrich) in 100 μl at 37 °C for 1 h, dilution 1:2000. The wells were washed 3 times and incubated at 37 °C for 30 min with 100 μl of fresh 0.4 mg/ml o-phenylenediamine and 0.4 mg/ml urea/H₂O₂ dissolved in 0.05 M Na₂HPO₄/0.05 M citric acid adjusted to pH 5. The color reaction was stopped with 50 μl of 1 M HCl per well, and the optical density measured after 1 h at 492 nm (OD₄₉₂) on an Infinite M200 microtiter plate reader (Tecan, Maennedorf, Switzerland). Results were normalized using beta-actin as control and presented as percent of expression.

Two different lot numbers of CXCL12/SDF-1 DuoSet were purchased from RnDsystems (catalog N° DY460). The kit consists of a mouse anti-human/mouse SDF1α capture antibody, a biotinylated goat anti-human/mouse SDF1α detection antibody, and a Streptavidin-horseradish-peroxidase-(HRP) conjugate.
Experiments were repeated with two other ELISA kits: the human SDF1α mini ELISA Development Kit (Peprotech; catalog N° 900-M92) which consists of an anti-human SDF1α capture antibody, a biotinylated anti-human SDF1α detection antibody and an avidin-HRP conjugate and the human SDF1α ELISA Kit (Raybiotech; catalog N° ELH-SDF1a) which includes a plate precoated with capture antibody, a biotinylated anti-human SDF1α detection antibody, and a streptavidin-HRP conjugate.
The analyses were performed in Nunc Maxisorp 96-well plates for the RnD and Peprotech kit and in the supplied plate for the Raybiotech kit, according to manufacturer’s instructions. The readout was performed in a Tecan Infinite M200 microtiter plate reader.