Precision microplate reader

Triacylglycerol (TAG), total cholesterol (TC), low-density
lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol
(HDL-C), glucose, alanine transferase (ALT), and aspartate aminotransferase
(AST) levels were analyzed using a precision microplate reader (Molecular
Devices Inc., New York, USA) following the protocol from the biochemical
assay kit (Sigma-Aldrich, Co. Ltd., USA).

Ninety-six-well ELISA plates were coated with 50 µl of purified PfMSA180-Tr4 protein diluted to 1 µg/ml in coating buffer (20 mM boric acid, pH 8.9) and incubated at 4 °C overnight. The plates were washed with phosphate-buffered saline with 0.1% (v/v) Tween-20 (PBS-T) and then blocked with 2 mg/ml of gelatin in coating buffer for 1 h at room temperature. Human serum samples were diluted to 1:600 in PBS-T, added to antigen-coated wells in duplicate, and incubated for 1 h at RT. After washing the plates were incubated with 1:3,000-diluted HRP-conjugated rabbit anti-human IgG (Dako Cytomation, Glostrup, Denmark) in PBS-T for 1 h at room temperature. The plates were again washed with PBS-T followed by incubation for 20 min at RT with 0.5 mg/ml azino-bis-3-ethylbenthiazoline-6-sulfonic acid (Wako Pure Chemical, Osaka, Japan) diluted in citrate buffer (0.1 M citric acid, pH 4.1). The reaction was stopped with 0.1 M citric acid, and optical densities (ODs) were determined at 415 nm using a precision microplate reader (Molecular Devices, Sunnyvale, CA). Antibody levels were measured in two independent assays.

A method to quantitate the subclasses of the meningococcal vaccine response has previously been described [19 (link)]. Briefly, serum or plasma samples and control standards (specific IgG1 and IgG3 antibodies against OMV) were applied to OMV-coated microtiter plates and incubated. Subclass specific monoclonal antibodies were obtained from the WHO/International Union of Immunological Societies (IUIS) Immunoglobulin Subcommittee, IgG1 (HP 6012), IgG2 (HP 6002), and IgG3 (HP 6050) [19 (link)]. The plates were read at 405 nm using a Precision microplate reader from Molecular Devices. The same principle was applied to detect the influenza and pneumococcal IgG subclass vaccine response, but as no subclass specific antibodies were available, only the amount of one IgG subclass compared to another could be determined.

Sera samples were serially diluted two-fold in PBS and serum antibody titers were determined by ELISA, as reported previously [32 (link)]. Briefly, 96-well ELISA plates were treated with coating buffer for 1 h and washed with PBST (0.05% Tween-20 in PBS). Then, purified recombinant Esa1 [29 (link)], dissolved in coating buffer (10 μg/ml), was added to the plates (100 μl per well). After incubation at 4°C overnight, the plates were washed with PBST, coated with 250 μl of 1% albumin from bovine serum (BSA) per well, and incubated at 22°C for 2 h. Afterward, the plates were washed three times and diluted serum was added to the plates at 100 μl/well. Then, the plates were incubated and washed, as described above. Rat anti-tongue sole IgM antibody (dilution, 1/500) was added to the plates, which were then incubated at 22°C for 1 h. The plates were washed three times, as described above, and horseradish peroxidase-conjugated goat anti-rat IgG (dilution, 1/1000; Beijing Biosynthesis Biotechnology Co., Ltd.) was added to each. The plates were then incubated at 22°C for 1 h. Color development was performed using the Soluble TMB Kit (Tiangen Biotech Co., Ltd.). The plates were read at 450 nm with a precision microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The assay was performed three times.

In order to evaluate the growth inhibitory effect of fisetin on different cancer cell lines, the colorimetric assay SRB (Sulforhodamine B assay) was used as previously described [27 (link),28 (link)]. Briefly, cells were cultured with or without gradually increasing doses of fisetin for 48 h. After medium aspiration, the cells were fixed, stained and incubated at room temperature for 30 min. After washing, tris-base solution (10 mM) was added to dissolve retained SRB dyes. Finally, absorbance was measured using a precision microplate reader (Molecular Devices, Sunnyvale, CA).