Commonly used tetrazolium dye reduction assays measuring NAD(P)H levels (MTT and CCK‐8) were employed as surrogate measures of cell proliferation/viability. 8 × 103 cells per well were seeded into 96 well plates. Cells were incubated with 10 μl of CCK8 reagent and incubated at 37°C until color development. Absorbance measurements were performed at 450 nm using a microplate reader (Thermo Scientific™ Multiskan™ FC). For MTT assays, 20 μl of MTT reagent at 5 mg/ml was added into each well for a final concentration of 0.5% and incubated for 4 h at 37°C. The medium was removed, and 100 μl 100% DMSO was added to each well followed by vigorous mixing. After incubation at room temperature for 10 min, samples were mixed, and absorbance was measured at 540 nm in a microplate reader (Thermo Scientific™ Multiskan™ FC).
Multiskan fc
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Finland, Japan, Germany, United Kingdom, France, Italy, India, Australia, Canada, Singapore, Denmark, Spain, Ireland, Switzerland
The Multiskan FC is a microplate photometer designed for absorbance measurements. It is capable of reading 6- to 96-well plates and can be used for a variety of applications, including enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and colorimetric assays.
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Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (101 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (80 mentions)
Cck 8, by Dojindo Laboratories (42 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (40 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (38 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (35 mentions)
Cck 8 assay, by Dojindo Laboratories (24 mentions)
Cck 8, by Beyotime (22 mentions)
96 well plate, by Corning (20 mentions)
Cell counting kit 8 (cck8), by Beyotime (19 mentions)
Bovine serum albumin (bsa), by Merck Group (19 mentions)
Cell counting kit 8 cck 8, by Beyotime (18 mentions)
Lmc 3000, by Biosan (16 mentions)
Cck 8, by Thermo Fisher Scientific (16 mentions)
Cck 8 solution, by Dojindo Laboratories (14 mentions)
Mtt solution, by Merck Group (14 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (14 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (14 mentions)
Microtiter plate, by Corning (13 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
1 728 protocols using multiskan fc
1
Colorimetric Assays for Cell Viability
2
Neuroprotective Effect of CDDO in PC-12 Cells
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PC-12 cells (BCRC 60048) were maintained in an RPMI-1640 medium (Gibco, Waltham, MA, USA) enriched with 10% horse serum and 5% fetal bovine serum (both from Gibco), in a 5% CO2 humidified environment at 37 °C. To induce a neuronal phenotype, these cells were treated for 14 days with 50 ng/mL of nerve growth factor on culture flasks coated with Collagen IV. For experiments, PC-12 cells (1 × 104 cells per well) were plated in 24-well plates and exposed to 500 μM H2O2. Concurrently, they were treated with varying concentrations of CDDO (ranging from 0 to 30 μg/mL) for 24 h. Following treatment, the culture medium was discarded. An MTT reagent was then added to each well at a concentration of 0.5 mg/mL and the cells were incubated for an additional 90 min. After this incubation, the medium was removed, and the purple formazan precipitate was solubilized with 200 μL of DMSO per well. Absorbance was measured using a Multiskan FC microplate photometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA).
3
Cytotoxicity Assessment via WST-1 and LDH Assays
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Cell viability was assessed by water-soluble tetrazolium 1 (WST-1) assay. The Cell viability was checked using EZ-Cytox water-soluble tetrazolium salt (WST) assay kit (EZ-3000; Dogenbio, Seoul, Republic of Korea). The absorbance was measured at 450 nm by microplate reader (Multiskan FC; Thermo Fisher Scientific). Cytotoxicity was evaluated by using Lactate dehydrogenase (LDH) Cytotoxicity Detection Kit (MK401; TAKARA, Otsu, Japan) according to the manufacturer’s protocols. Briefly, a total of 100 μL of LDH reaction reagent was added into 100 μL of supernatants and then incubated at 37 °C for 30 min. Absorbance was determined at 450 nm using microplate reader (Multiskan FC; Thermo Fisher Scientific).
4
Evaluating HACB/BCNU Nanoparticle Cytotoxicity
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The cytotoxicity of HACB/BCNU NPs was investigated by an MTT assay. Generally, HeLa cells were seeded in 96-well plates at a density of 5 × 103/well and cultured for 24 h at 37 ℃. Then, the cells were treated with BCNU and HACB/BCNU NPs (at equivalent BCNU concentrations of 10, 20, 50, 100, 200, 400, 600 and 1000 µM) under normoxic and hypoxic conditions. For the combination-treated groups, the cells were pretreated with 40 µM O6-BG for 2 h before exposure to BCNU. Moreover, HeLa cells and a normal cell line b End.3 cells were treated with HACB NPs (0.02, 0.04, 0.10, 0.21, 0.42, 0.83, 1.25 and 2.08 mg/mL) to evaluate the cytotoxicity of blank micelles. After a 24-h treatment, 10 µL MTT solutions (5 mg/mL) were added in each well, and the cells were incubated in the dark for 4 h. Subsequently, the medium was removed and formazan crystals were dissolved in 150 µL DMSO. Finally, the absorbance at 560 nm was determined by a Thermo Scientific Multiskan FC (Multiskan FC, Thermo Scientific, Waltham, MA, USA). The cell viability was calculated according to Eq. (4 ) as follows:
Here, the is the absorbance values of the drug-treated cells, is the absorbance values of blank only containing medium and is the absorbance values of the cells without drug treatment.
Here, the is the absorbance values of the drug-treated cells, is the absorbance values of blank only containing medium and is the absorbance values of the cells without drug treatment.
5
Evaluating PDLSC Growth on Nanofibers
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To evaluate the cellular growth performance of PDLSCs treated with DAE, cells from passages 3 to 5 were seeded into 96-well plates at 1 × 104 cells/well in the cell culture medium described above, which was replaced every three days for seven days. This is a colorimetric assay was based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) for assessing cell metabolic activity as an indicator for cell viability, proliferation, and cytotoxicity. The well containing the proliferated cells was treated with 5 mg/mL MTT at 37 °C for 4 h. The cultivated medium was removed, and formazan was solubilized in DMSO. The metabolized MTT was measured based on optical density at 570 nm using a spectrophotometer (Multiskan FC, Thermo Fisher Scientific). The nanofiber is clamped flat with a microcentrifuge tube and cut to form a culture well, then sterilized with autoclave. PDLSCs from passages 3 to 5 were seeded into the nanofiber at 1×104 cells/well. The cellular viability of PDLSCs on nanofibers was measured using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) for 21 days. The proliferated cells on nanofibers were treated with 10% CCK-8 reagent at 37 °C for 4 h, and then optical density at 450 nm was measured using a spectrophotometer (Multiskan FC, Thermo Fisher Scientific).
6
Quantifying Tau and NfL in Cell Media
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Medium was collected after 48 hours of incubation with cells, centrifuged at 400 × g for 5 minutes to remove cell debris and then frozen in −80 °C until analysis. Total tau concentration in cell media was measured using INNOTEST® hTAU Ag ELISA (Fujirebio) according to the manufacturer’s instructions. The plate was mixed and the absorbance was quantified at 450 nm at a Multiskan FC (Thermo Scientific) within 15 min. NfL concentration in the cell media was measured using NF-light® (Neurofilament light) ELISA from UmanDiagnostics, according to the manufacturer’s instructions. The absorbance was quantified at 450 nm with 620 nm reference wavelength at a Multiskan FC (Thermo Scientific).
7
Antioxidant Enzyme Activity Assay
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According to the ratio of tissue quality (g): extract volume (ml) 1:5-10, the fresh weight of leaf and root tip was 0.1g, respectively. After centrifugation with phosphate buffer solution of pH = 7.0 at 4 °C, the supernatant was taken as the follow-up determination sample. Each set of 3 biological repeats contained 5 technical repeats. SOD, CAT, POD, and MDA were determined (Suzhou Keming Biotechnology Co., Ltd. kit) by Microplate reader (Multiskan FC, Thermo fisher scientific, Waltham, MA, USA). The protein concentration was determined (the BCA method) by Microplate reader (Multiskan FC, Thermo fisher scientific, Waltham, MA, USA).
8
ELISA Protocol for Antibody Detection
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To test the rabbit anti-oPRL polyclonal antibodies, 96-well plates were coated with 0.1 μg per well of oPRL in PBS (PH 7.2) overnight at 4°C. The plates were subsequently washed four times with PBS in 0.05% Tween 20 (PBST), blocked with 2.5% non-fat powder in PBS and incubated with Rabbit antisera diluted to their optimum concentration in PBST. The plates were incubated for 1.5 h at 37°C, washed and reincubated with the HRP-conjugated goat anti-rabbit IgG. After 1 h, the plates were washed and incubated with the tetramethylbenzidine (TMB) substrate. The results were read at 450 nm in an automatic ELISA plate reader (Multiskan FC, Thermo Fisher Scientific Inc., Waltham, MA, USA).
To detect the monoclonal anti-idiotypic antibodies to PRL, 96-well plates were coated with purified anti-PRLR F(ab′)2 fragments or with F(ab′)2 fragments of control antibodies overnight at 4°C. The plates were then washed four times with PBS in 0.05% Tween 20 (PBST), blocked with 2.5% non-fat powdered in PBS and incubated with the culture supernatants from the hybridomas, and the wells were incubated at 37°C for 1 h. After washing, goat anti-mouse IgG-FC HRP secondary antibody was added, and after 1 h, the plates were washed and incubated with TMB substrate, and the results were read at 450 nm in an automatic ELISA plate reader (Multiskan FC, Thermo Fisher Scientific Inc., USA).
To detect the monoclonal anti-idiotypic antibodies to PRL, 96-well plates were coated with purified anti-PRLR F(ab′)2 fragments or with F(ab′)2 fragments of control antibodies overnight at 4°C. The plates were then washed four times with PBS in 0.05% Tween 20 (PBST), blocked with 2.5% non-fat powdered in PBS and incubated with the culture supernatants from the hybridomas, and the wells were incubated at 37°C for 1 h. After washing, goat anti-mouse IgG-FC HRP secondary antibody was added, and after 1 h, the plates were washed and incubated with TMB substrate, and the results were read at 450 nm in an automatic ELISA plate reader (Multiskan FC, Thermo Fisher Scientific Inc., USA).
9
Quantification of HGPRT and XO Activities
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HGPRT activity was measured by quantifying the rate of IMP production, which is oxidized by IMPDH, using the PIERCE HPRT Assay Kit (NOVOCIB, Lyon, France) following the manufacturer's instructions. Briefly, enzymatic assay was performed in reaction buffer (1X) containing IMPDH, DTT, NAD + and PRPP. The reaction mixture was recorded every 5 min for 120 min at 340 nm using a microplate reader (Multiskan FC, Thermo Scientific, Finland), and HGPRT activity expressed as nmol mg -1 protein h -1 .
Xanthine oxidase (XO, E.C. 1.17.3.2) activity XO activity was measured using an Amplex® Red Xanthine/Xanthine oxidase assay kit (A22182; Invitrogen, Paisley, UK). The quantification is based on the superoxide anions (O 2 •-) produced by XO, which are degraded to hydrogen peroxide (H 2 O 2 ), which reacts stoichiometrically with Amplex® Red reagent to generate the oxidation product, resofurin. Briefly, the enzymatic assay was performed in reaction buffer (1X) containing 100 µmol L -1 Amplex Red reagent solution, 0.4 U mL -1 HRP and 200 µmol L -1 hypoxanthine, incubated for 30 min at 37°C. The presence of resofurin was recorded at 550 nm using a microplate reader (Multiskan FC, Thermo Scientific, Finland), and the XO activity expressed as mU mg -1 . One unit of XO activity is defined as the amount of enzyme required to produce 1 µM of UA from HX per min at 25°C.
Xanthine oxidase (XO, E.C. 1.17.3.2) activity XO activity was measured using an Amplex® Red Xanthine/Xanthine oxidase assay kit (A22182; Invitrogen, Paisley, UK). The quantification is based on the superoxide anions (O 2 •-) produced by XO, which are degraded to hydrogen peroxide (H 2 O 2 ), which reacts stoichiometrically with Amplex® Red reagent to generate the oxidation product, resofurin. Briefly, the enzymatic assay was performed in reaction buffer (1X) containing 100 µmol L -1 Amplex Red reagent solution, 0.4 U mL -1 HRP and 200 µmol L -1 hypoxanthine, incubated for 30 min at 37°C. The presence of resofurin was recorded at 550 nm using a microplate reader (Multiskan FC, Thermo Scientific, Finland), and the XO activity expressed as mU mg -1 . One unit of XO activity is defined as the amount of enzyme required to produce 1 µM of UA from HX per min at 25°C.
10
Cytotoxicity Assessment of Nanoparticles
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To assess the cytotoxic effect of nanoparticles, an MTT test was performed. For the test, AgNPs with a final concentration of 0.05–125 µg/mL was added into a pre-seeded with cancer cells 96-well plate. Cells without exposure to AgNPs were used as a negative control. Cells incubated in a medium supplemented with 0.3% hydrogen peroxide were used as a positive control (dead cells). After 24 h of incubation, the medium from all wells was aspirated, and replaced with the fresh medium of the same composition containing 0.5 mg/mL MTT reagent (Paneco, Russia) was added and kept for 4 h at 37 °C in a CO2 incubator. The optical density was measured on a spectrophotometer (Multiskan FC, TermoFisher, Waltham, MA, USA) at a wavelength of 570 nm. The calculation was performed by subtracting the optical density of the background and the optical density of the positive (dead cells) control. Calculation of cell viability after exposure was performed as a percentage of alive cells in the experiment towards the viability control (cells without exposure to AgNPs).
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