Briefly, 5 × 103 cells/well were inoculated into a 96-well plate and cultured for 24 h, then transfected with siRNA. Cell proliferation was assessed by using Cell Counting Kit-8, CCK-8 (Beyotime Institute of Biotechnology, Shanghai, China). Finally, absorbance was measured after 24-, 48- and 72 hours transfection by an ELISA microplate reader (Bio-Rad, Hercules, CA, USA). Ethynyl deoxyuridine (Edu) assay was performed by using an Edu Kit (Ribobio, Guangzhou, China) following the manufacturer's instructions. Experiments were repeated five times.
Elisa microplate reader
Manufactured by Bio-Rad
Sourced in United States, Japan, Switzerland
The ELISA microplate reader is a laboratory instrument used to measure the absorbance or fluorescence of samples in a microplate format. It performs quantitative analysis of enzyme-linked immunosorbent assays (ELISA), a widely used technique for the detection and quantification of proteins, antibodies, hormones, or other analytes.
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Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (18 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Cell counting kit 8 (cck8), by Beyotime (5 mentions)
Mtt solution, by Merck Group (4 mentions)
96 well plate, by Thermo Fisher Scientific (4 mentions)
Cell counting kit 8 (cck8), by Transgene (3 mentions)
Cck 8, by Dojindo Laboratories (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Peroxidase streptavidin goat anti mouse iga hrp, by Southern Biotech (2 mentions)
Cck 8, by Beyotime (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Tween 20, by Merck Group (1 mentions)
Tetramethylbenzidine, by Merck Group (1 mentions)
1 step ultra tmb, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Bovine serum albumin (bsa), by R&D Systems (1 mentions)
Bovine serum albumin (bsa), by ICN Biomedicals (1 mentions)
Avidin biotinylated horseradish peroxidase complex, by Vector Laboratories (1 mentions)
Recombinant mouse cst6, by R&D Systems (1 mentions)
148 protocols using elisa microplate reader
1
Cell Proliferation Assays with siRNA
2
Seroprevalence of Toxoplasmosis in Pregnancy
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A total of 480 peripheral blood samples were collected from women with a spontaneous abortion at <20 weeks of gestation, and 200 peripheral blood samples were collected from women with a normal delivery at a gestational age of 38-39 weeks. The participants in this study were in the age range of 14-53 years. The serological evidence of toxoplasmosis was investigated by detecting T. gondii IgM and IgG antibodies. The serum samples were separated and stored in aliquots at −20°C until further analysis. Next, they were tested for the presence of IgM and IgG antibodies, using an ELISA-based NovaLisa test kit. Moreover, a Toxoplasma-specific IgG avidity assay was performed using an avidity T. gondii IgG ELISA kit (NovaTec GmbH, Germany) to differentiate between acute and chronic infections. All samples were tested according to the manufacturer's instructions. The results were then read by an ELISA microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) and compared with the calibrator and the controls.
3
Cell Growth Analysis by CCK-8 Assay
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The effects of YAP/Mask2 complex on cell growth were determined by Cell Counting Kit-8 assay (Transgene, China). Briefly, 5×103 cells per well were seeded in a 96-well plate for 12 hours and then transfected as mentioned above. After 48 hours of transfection, 100μl fresh medium containing 10% of CCK-8 was replaced to each well and the cells were cultured for another 1 hour. The absorbance at 450 nm was determined using an ELISA microplate reader (Bio-Rad). Experiments were repeated for three times.
4
Quantifying Dog Ig Isotypes by ELISA
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Immunoglobulin (Ig) isotypes A and M were quantified by indirect ELISA using mouse anti-dog IgA horseradish peroxidase (HPR) conjugate and mouse anti-dog IgM HPR as primary antibodies, and goat anti-mouse IgG-HPR, Novex) as a secondary antibody. The Mucosal Immunology Laboratory of the Veterinary School at Bristol University, UK provided the primary antibodies. For details on the protocols used see Supporting Information. IgG concentrations were measured with a protein A ELISA as reported previously [37 (link)], with slight modifications (see Supporting Information). Absorbance was measured in an ELISA microplate reader (BioRad, USA) at 450 nm. For each isotype, absorbance readings were interpolated on a standard curve using dog serum (Bethyl Laboratories, USA) as a reference. All reactions were run in triplicate.
5
Cytotoxicity Evaluation of PβAE-447 Polymers
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The relative cytotoxicity of pure and PEGylated PβAE-447 was separately investigated by using the 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Human embryonic kidney (HEK-293), bronchial epithelial (BEAS-2B), and lung adenocarcinoma epithelial (A549) cells were incubated in 96-well plates with DMEM (100 μL) for 24 h. When the cells achieved almost 70% confluence, the old media was removed. The new media containing various concentrations of pure and PEGylated PβAE-447 were added to each well. The 96 well plates were incubated for 24 h, and then 25 μL MTT solution (5.0 mg/ml) in PBS was added to each well. After 4 h of incubation, the MTT solution and DMEM were aspirated, and 150 μL DMSO was added to dissolve the formazan crystals. The plates were placed on a shaker for 10 min before recording the absorbance at 570 nm by an ELISA microplate reader (Bio-Rad, California, United States). The percentage of cell viability was calculated by using Eq. (3) where Asample is the absorbance from the treated cells and Acontrol is the absorbance from untreated cells.
Moreover, the cell viability of PEI 25 KDa was also determined.
Moreover, the cell viability of PEI 25 KDa was also determined.
6
Assessing Breast Cancer Cell Proliferation
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Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, the proliferation of BC cells was examined. BC cells were infused into 96-well plates at a density of 3 × 103 cells per well 72 h after transfection. By pouring 20 L of a 5 mg/mL MTT solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) into each well, cell proliferation was assessed 72 h after seeding. For an additional 4 h, the plates were incubated at 37 °C in a humidified environment composed of 95% air and 5% CO2. Dimethyl sulfoxide (150 L) was used in place of the culture medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Finally, an ELISA microplate reader was used to measure the absorbance at a wavelength of 490 nm (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
7
Cytotoxicity Evaluation of MPEG-PCL-CS Nanosuspension
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The MTT assay was employed to evaluate the in vitro cytotoxicity of the MPEG-PCL-CS nanosuspension against HCEC, HLEC, and L-929 cells. Briefly, cells were seeded at a density of 1 × 104 cells/well in 96-well plates and incubated for 24 h in a 5% CO2 incubator. Then, aliquots of the MPEG-PCL-CS nanosuspension ranging in concentration from 10 to 5000 μg/mL were added to the wells, followed by incubation for another 24 h. Untreated cells in growth medium were used as controls. After 24 h of incubation, 20 μL of MTT solution (5 mg/mL in phosphate buffered saline (PBS)) was added to each well, and the plates were incubated for an additional 2 h. The MTT solution was then carefully removed and DMSO was added. The absorbance values were recorded using an ELISA microplate reader (Bio-Rad, Hercules, CA) at 570 nm. Cell viability (%) was calculated according to the following equation: cell viability (%) = absorbance test/absorbance control × 100%, where the absorbance control was the absorbance for the control wells. All data are expressed as the mean of six measurements (mean ± SD, n = 6).
8
Evaluating Cytotoxicity of Zeolite Compound
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To assess the toxic potential of the MZC, we performed a cell cytotoxicity assay using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) technique on BIECs, prepared as previously indicated. The cells were seeded at a density of 105 cells per well in 96-well flat-bottom plates and allowed to adapt for 24 h under physiological growth conditions. The cultures were then exposed for 24 h to MZC at increasing concentrations, ranging from 19.5 μg/mL to 10 mg/mL. Cell cultures treated only with the medium were used as controls. After 24 h, the cells treated with zeolite were incubated for 2 h with 150 μL of MTT. The MTT assay involves the conversion of the water-soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to insoluble formazan. The formazan was solubilized with 100 μL DMSO, and the absorbance of each sample was read at 490 nm using an ELISA microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability (%) was calculated by using the equation: cell viability (%) = OD test × 100/OD control. The experiment was carried out in triplicate.
9
Anti-L. major IgG Quantification by ELISA
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The sera of mice were collected three weeks after the last vaccination and seven weeks after the challenge. Prior to usage, sera were kept at −20°C. To detect the levels of anti-L. major Ig G, the sera were tested with ELISA kits. The 96 wells-plates were coated at 4°C overnight with soluble L. major antigens at concentration of 10 μg/ml prepared in 100 mM carbonate-bicarbonate buffer (pH=9.6). Blocking was carried out with 5% dried skimmed milk in PBS (pH=7.2) for 2 hr at the room temperature. After washing with PBS containing 0.05% Tween 20 (Merck KGaA, Darmstadt, Germany) (PBST20), the sera were diluted 1/100 in 5% dried skimmed milk-PBST20 (100 lL per well) and incubated for 2 hr at 37°C. After washing, the bound antibodies were detected by incubation for 2 hr at 37°C with anti-mouse IgG conjugated with peroxidase (DA-KO, Denmark) at 1/2000 dilution in 5% dried skimmed milk-PBST20. Peroxidase activity was revealed by adding 100 μl per well of tetra methyl benzidine (Sigma-Aldrich) in dark. The reaction was stopped after adding 100 μl of sulfuric acid and Optical Density (OD) was read at 450 nm by the ELISA micro plate reader (Bio-Rad, USA) 22 .
10
Quantification of Mouse and Human Cystatin E/M
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Recombinant mouse Cst6 and human CST6 (both from R&D Systems, Minneapolis, MN, USA) were used as a standard and as a negative control, respectively, in concentrations varying from 5 to 0.156 ng/ml. The wells of a 96-well plate were coated overnight at 4°C with polyclonal rabbit anti-human CST6 antibody (2 (link)), followed by incubation with 1% bovine serum albumin (ICN Biomedicals, Aurora, OH, USA) and 1% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS for 30 min. Subsequently, standards, controls, and samples (undiluted up to 32× diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30 min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min. The above incubation steps were performed at 37°C and separated by repeated washing steps with PBS/0.05% Tween-20. Chromogenic substrate 1-step Ultra TMB (Thermo Fisher Scientific) was used as substrate for detection and the reaction was stopped by adding 4 M H2SO4. Each well was measured for mouse Cst6 at an absorbance of 450 nm with an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).
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