Wallac 1420 arvosx multilabel counter

Cells were plated on 24-well plates at the density of 1 × 10⁵ cells/well and infected with SINV at a multiplicity of infection (MOI) of 1. Cells were also infected with UV-inactivated SINV at an MOI of 1 or 5. Cell viability was evaluated after 48 h. Each plate was equilibrated to room temperature (approximately 25 °C) for 20 min followed by the addition of 200 μL CellTiter Glo reagent (Promega, Madison, WI, USA) to each well. Plates were gently agitated on a plate shaker for 1 min and incubated for an additional 10 min at room temperature followed by immediate evaluation of cell viability by measuring the luminescence using a plate reader (Wallac 1420 ARVOsx Multilabel Counter, Perkin-Elmer, Japan). The cell viability of mock infection was considered 100%. These data were presented as mean ± standard deviation (SD) of three determinations.

A soft agar colony formation assay was carried out using the CytoSelect 96-well Cell Transformation Assay kit (Cell Biolabs) as described previously^{13 (link)}. Cells were cultured for 30 days. Colonies were lysed and quantified with CyQuant GR dye using a fluorometer equipped with a 485/520 nm filter set (Wallac 1420 ARVOsx multilabel counter, PerkinElmer).

HSY cells were loaded with fura-2 by incubation for 20 min at 37°C with 3 μM fura-2-acetoxymethyl ester (Dojindo, Kumamoto, Japan) suspended in BSS-BSA, rinsed twice, resuspended in 5 mL of BSS-BSA, and stored at 4°C. Fura-2-loaded HSY cells were transferred to a 96-well black plate (Biomedical Science, Tokyo, Japan) and alternately illuminated by excitation at 340 and 380 nm. A microplate reader (Wallac 1420 ARVO SX Multilabel Counter, Perkin Elmer Co., Ltd., Massachusetts, USA) was used for measurements. Prior to measurements, cells were incubated at 37°C for 5 min and pre-treated with quercetin (1 μM, 10 μM, 100 μM) for 2 minutes. Subsequently, carbachol (cch) (30 μM or 100 μM) was added directly to the cell suspension during fluorescence measurements. The results are expressed as the fluorescence ratio (F340/F380). Fold changes of intracellular Ca²⁺ concentration were calculated using the values before and after cch injection.

BALB/c mice were used to evaluate PCA response according to previously described protocols [35 (link),36 (link)] with some modification. Briefly, anti-DNP IgE (150 ng/10 μl; Sigma) was injected intradermally in the right ear. As a blank, PBS was injected intradermally in the left ear. To explore the effect of histone H1 on PCA response, calf thymus histone H1 (5 μg/10 μl; Merck Millipore) was injected intradermally both in the left and right ears (n = 3). As a sham control (n = 8), PBS was injected intradermally in both right and left ears. Twenty-three hrs later, 200 μl of PBS (vehicle control; n = 7), 100 μg of isotype IgG1 (n = 8) or SSV mAb (n = 7) was injected intravenously via tail vein. One hr later, 200 μg of DNP-HSA (Sigma) in 100 μl of evans blue solution (0.5 or 1%; Tokyo Chemical Industry, Co., Ltd., Tokyo, Japan) was injected intravenously to induce anaphylaxis. After 30 minutes, PCA response was evaluated by the measurement of evans blue dye in the ears. Evans blue dye in the ears was extracted by overnight incubation with formamide at 63°C, and the absorbance at 595 nm was determined using a Wallac 1420 ARVOsx Multilabel Counter (PerkinElmer). This study was performed in Hiroshima University using protocols reviewed and approved by the under the Committee on Animal Experimentation of Hiroshima University.

H9C2 cells, primary cultured cardiomyocytes, MDA-MB-231 cells, H1299 cells, and HT29 cells were washed with serum free medium and added to Counting Kit-8 medium (Dojindo, Kumamoto, Japan) for 1 h. After incubation, measurement of absorbance was performed using a Wallac 1420 Arvo Sx multilabel counter (Perkin Elmer, Waltham, MA, USA). The relative percentage of cell survival was calculated by dividing the absorbance of the treated cells by that of the control in each experiment.

Cell-free culture supernatants were collected from RuV-infected cells at the indicated times after RuV infection. According to the manufacturer’s instructions, the amount of human IFN-β was quantified using the VeriKine human IFN-β ELISA kit (PBL Assay Science, Piscataway, NJ, USA, 41410). The absorbance of test samples was measured using the Wallac 1420 ARVOsx multi-label counter (Perkin Elmer, Waltham, MA, USA) and converted to picograms per milliliter using a standard curve generated by serially diluting the standard in the sample plate. Indicated data are representative of two experiments.