Cells were seeded in 96-well plates at a density of 3000 cells/well. After treatment, the cell culture medium was removed and replaced with 90 μL serum-free 1640 medium. Cell Counting Kit-8 (CCK8, Dojindo, Japan) staining solution (10 μL) was added to the cells and incubated at 37 ℃ for 2 h, then absorbance at 450 nm was measured by Absorbance Reader (Tecan, Switzerland).
Absorbance reader
Manufactured by Tecan
Sourced in Switzerland, Austria
The Absorbance reader is a laboratory instrument designed to measure the absorbance of light by a sample. It is used to quantify the amount of light absorbed by a substance in a sample, which can be used to determine the concentration of that substance.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Mouse cd14, by R&D Systems (1 mentions)
Biotinylated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Streptavidin horse radish peroxidase conjugate, by Zymo Research (1 mentions)
Protein g sepharose 4 fast flow, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
High binding 96 well elisa plates, by Corning (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti human igg antibody, by Jackson ImmunoResearch (1 mentions)
Bovine serum albumin (bsa), by Jackson ImmunoResearch (1 mentions)
Cytotox 96 non radioactive cytotoxicity assay ldh, by Promega (1 mentions)
Prism 4, by GraphPad (1 mentions)
Microsoft excel, by GraphPad (1 mentions)
Elisa plate, by Greiner (1 mentions)
Goat anti mouse igg, by Dianova (1 mentions)
13 protocols using absorbance reader
1
Cell Viability Assay Protocol
2
Cytokine ELISA Quantification
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The supernatants were collected by centrifugation after stimulation of the cultured cells. Human and mouse CCL20 (R&D Systems), mouse CD14 (R&D Systems), and human BD-2 (Arigo Biolaboratories) ELISAs were performed according to manufacturer's instructions. Absorbance was measured in absorbance reader (Tecan).
3
ELISA for Crude OV Adult ES Antigen
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Plates were washed 5 times with PBST, and 100 μL of a protein A purified rabbit IgG (10 μg/mL) against crude OV adult ES antigen extract was added to each well and incubated at 37°C for 2 hours. After washing three times, 1:4,000 diluted biotinylated-goat anti rabbit IgG (Invitrogen, CA, USA) in PBST was added and incubated at 37°C for 1 hour. Thereafter, the plates were washed 3 times with PBST, and 100 μL/well of streptavidin horseradish peroxidase (HRP) conjugate (Zymed, CA, USA) was added diluted 1:10,000 in PBST. After incubation for 30 min, the plates were washed 3X with PBST, and the substrate solution Orthophenylenediamine hydrochloride (Sigma, St. Louis, MO, USA) was added to wells and incubated for 20 min in the dark at room temperature. The reaction was stopped by the addition of 2M sulfuric acid (H2SO4), and the plates were read on an absorbance reader (Tecan, Austria) at the optical density (OD) at 492 nm.
Two well-trained laboratory personnel were responsible for execution of the sample analysis of both tested samples (index cases) and reference standard tests and they were analyzed simultaneously. During the sample execution, the sample IDs were blinded and the laboratory staffs had no knowledge of the sample subjects.
Two well-trained laboratory personnel were responsible for execution of the sample analysis of both tested samples (index cases) and reference standard tests and they were analyzed simultaneously. During the sample execution, the sample IDs were blinded and the laboratory staffs had no knowledge of the sample subjects.
4
SARS-CoV-2 Spike Protein IgG ELISA
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For analysis of IgG interaction with SARS‐CoV‐2 protein, high binding 96‐well ELISA plates (Corning Inc., Corning, NY, USA) were coated with SARS‐CoV‐2 spike protein (5 µg/ml) at 4°C overnight, washed 3× with PBS and blocked with PBS, containing 5% BSA (Carl Roth, Karlsruhe, Germany) for 60 min at RT. Thereafter, IgGs were tested at 3‐fold dilutions (1:2) starting at concentrations of 166 µg/ml in PBS / 5% BSA for 120 min at RT. IgGs were isolated with Protein G Sepharose® 4 Fast Flow (GE Healthcare). The plates were washed 3× and incubated with horseradish peroxidase‐conjugated goat anti‐human IgG antibody (Jackson ImmunoResearch West Grove, PA, USA) (1:2500 in PBS/5% BSA) for 60 min at RT. ELISAs were developed using 2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) solution (ABTS, Thermo Fisher Scientific), and absorbance (OD 415–695 nm) was measured with absorbance reader (Tecan, Männedorf, Switzerland).
5
Cytotoxicity Assay for Cancer Cells
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HCT116 or SW480 cells were seeded in triplicate at a density of 1 × 104cells per well into 96-well plates. After 24-hour incubation in complete RPMI 1640 with 10% FBS, cells were exposed to Cisplatin or Doxorubicin at different concentrations. Upon measurement, RPMI 1640 medium and CCK8(Cell Counting Kit-8) staining solution was added to cells at each well for 2 hours at 37 °C. The absorbance was measured at 450 nm daily using an absorbance reader (TECAN, Switzerland). The percentage of cell survival was then calculated.
6
Cell Proliferation Assay using CCK8
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Cell Counting Kit-8 (CCK8) (CK04, Dojindo) assays were performed to evaluate cell proliferation ability. Cells were seeded into 96-well plates at a density of 3 × 103 cells/well. Subsequently, the medium was replaced with 90 μl basic RPIM 1640 medium and 10 μl CCK8. After incubation at 37 °C for 2 h, the absorbance of each well at 450 nm was measured by Absorbance Reader (TECAN, Switzerland).
7
Cell Viability Assay for hCPCs
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A cell viability assay (WST kit, Ez-Cytox, Daillab, Seoul, Republic of Korea) was performed to compare the viability of the control and high glucose-exposed hCPCs. hCPCs were seeded on 96-well plates, and then the maintenance medium was changed to hCPC culture medium containing 5 mM, 15 mM, or 25 mM d -glucose. Culture plates were incubated for 24 h or 72 h. Following incubation, the d -glucose-containing culture medium was removed and replaced with WST solution. The plates were incubated for another 2 h, and the absorbance of each sample was measured at a wavelength of 450 nm using an absorbance reader (Tecan, Grodig, Austria).
8
Cytotoxicity Assessment of h-BN_AuNP Nanocomposite
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Cytocompatibility of the h-BN_AuNP nanocomposite was evaluated using the LDH CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). The LDH CytoTox 96® Non-Radioactive Cytotoxicity Assay measures lactate dehydrogenase released due to cellular membrane damage. The amount of formazan converted from tetrazolium salt is proportional to the number of lysed cells. The LDH assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA) and the absorbance was measured at 490 nm using a microplate spectrophotometer (Absorbance Reader, Tecan, Männedorf, Switzerland). The interaction between the solution with the nanocomposite in the cell culture medium and LDH assay components was tested in the absence of cells. The percentage of LDH released after 24, 48 and 72-h exposure was calculated using the Formula (3):
where A is absorbance.
where A is absorbance.
9
Quantifying H398 Plasma Concentrations
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To determine H398 plasma concentrations, ELISA plates (Greiner, Frickenhausen, Germany) were coated with huTNFR1-Fc fusion protein at 1μg/ml in PBS and incubated at 4°C overnight. Residual binding sites were blocked with 2% skim milk powder in PBS for 2 hours. Then, plasma was added for 2 hours and bound proteins were detected by incubation with HRP-conjugated anti-mouse IgG for 1 hour followed by incubation with TMB substrate solution. Reaction was stopped by addition of 1M H2SO4 and the absorbance at 450nm was determined with an absorbance reader (Tecan, Männedorf, Switzerland) and data were analysed using the software Microsoft Excel and GraphPad Prism 4 (GraphPad, La Jolla, CA).
10
MAIGA Protocol for IgG Quantification
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The original MAIGA protocol 19 was modified. In brief, 3 × 10 5 paraformaldehyde-fixed 293F cells were sensitized with 50 μL serum, washed, incubated with 10 μL capture moab (20 μg/mL), and lysed afterwards in 100 μL lysis buffer (2.4 g/L Tris, 8.76 g/L NaCl, 9.5 mL/L Triton X-100, and 1.86 g/L EDTA; pH 7.4) Cell lysates were immobilized onto a microtiter plate precoated with goat anti-mouse IgG. Bound human IgG was then detected with horseradish peroxidase-labeled goat anti-human IgG (Dianova), followed by incubation with the enzyme substrate (o-phenylenediamine) and colorimetric determination in an absorbance reader (Tecan) at 492 nm. The cut-off was defined as the twofold absorbance obtained with mock-transfected 293F cells.
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