Ez read 400 microplate reader

MCF7 proliferation of untransfected, EGFP-C1 transfected, and DCUN1D5-GFP transfected cells was evaluated after 24 h upon transfection in a 96-well plate by fixing cells with 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA, Cat: P6148) and staining it with crystal violet (Gibco, Grand Island, NY, USA, Cat: 42555) solution (0.1% in 20% methanol). For quantification, stained crystal violet was solubilized with 10% acetic acid. Absorbance was read at 620 nm (two measurements) with an EZ Read 400 microplate reader (Biochrom, Cambridge, UK).

The cytotoxicity of PEITC on PMA‐activated THP‐1 macrophages was measured using the MTT assay. The cells were seeded at a concentration of 1 × 10⁶ cells/well in 24‐well plates and treated with PEITC for 48 hr and treated with LPS (500 ng/ml) for 6 hr prior to harvest, and MTT solution (100 μL; 1 mg/ml) was added and incubated for a further 2 hr. The precipitated formazan was solubilized in 1 ml of 100% dimethyl sulfoxide. Finally, plates were placed in an EZRead 400 microplate reader (Biochrom) to measure absorbance at 570 nm.

Serum amyloid A (SAA) concentration in chicken sera was measured using a commercial ELISA kit: chicken serum amyloid A ELISA (Life diagnostics Inc., West Chester, PA, USA). According to the manufacturer’s instructions, ELISA was performed in duplicate for each standard and diluted serum sample. Absorbances were read at 450 nm using a Biochrom EZ Read 400 Microplate Reader (Biochrom, Cambridge, UK), and the concentrations were determined from the standard curve by a four-parameter logistic regression.

Cellular cytotoxicity was assessed by detecting LDH in neutrophil supernatants stimulated with RSV (10²–106 PFU/mL) for 180 minutes using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit according to manufacturer’s instructions. Readings were carried out at 490 nm wavelength, using EZ Read 400 microplate reader (Biochrom).

Mice sera were collected from all groups 4 weeks after prime, 1st boost immunization, and 2nd boost immunization using a retro-orbital plexus puncture. Sera from naïve mice were used as a negative control. Antibody responses against P. berghei antigen, AMA1 rVV, and MIC rVV were determined by enzyme-linked immunosorbent assay (ELISA), as described previously [17 (link)]. Briefly, 96-well immunoplates were coated with 100 μL of P. berghei antigen, AMA1 rVV, or MIC rVV at a final concentration of 2, 0.5, and 0.5 μg/mL, each respectively in 0.05 M, pH 9.6 carbonate bicarbonate buffer per well at 4 °C overnight. Afterwards, 100 μL of serially diluted serum samples were added into the respective wells and incubated at 37 °C for 1.5 h. HRP-conjugated goat anti-mouse IgG (100 μL/well, diluted 1:2000 in PBS) was used to determine the P. berghei-specific IgG antibody response. O-phenylenediamine was dissolved in citrate substrate buffer, and OD₄₉₂ was measured using an EZ Read 400 microplate reader (Biochrom Ltd., Cambridge, UK).

Samples were diluted at 1/10 in the diluent provided in the ELISA kit of interest (Mybiosource). The procedure was performed according to the instructions of the kits. Absorbances were measured at 450 nm using the EZ read 400 microplate reader (Biochrom).