Caco-2 cells were plated at a cell density of 4 × 105 cells per well in 96-well plates and incubated at 37 ± 1 °C in an atmosphere of 5% CO2. After 24 h of culture, the medium was replaced with SNAC, R1 or SA. After 24 h, the medium was discarded and the wells were washed twice with hanks balanced salt solutions (HBSS). 200 μL MTT solution (0.5 mg·mL−1 in PBS) was added to each well, and incubated 4 h at 37 ± 1 °C for MTT formazan formation. Subsequently, the supernatant was carefully removed and the wells were washed twice with PBS. DMSO (200 μL) was added to each well and the plates were then mildly shaken for 15 min to ensure the dissolution of formazan crystals. The optical density values were measured by using MQX200 microplate reader (Bio-Tek, Shoreline, WA, USA) at wavelength 570 nm. Six replicates were read for each sample and mean value was used as the final result. The spectrophotometer was calibrated to zero absorbance using culture medium without cells.
Mqx200 microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The MQX200 microplate reader is a versatile laboratory instrument designed for a wide range of absorbance-based assays. It is capable of reading 96-well microplates with a wavelength range of 200-999 nm. The MQX200 provides accurate and reliable data for a variety of applications, including enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and colorimetric assays.
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7 protocols using mqx200 microplate reader
1
Caco-2 Cell Viability Assay
2
Serum Cytokine Quantification via ELISA
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The content of IL-6, 12, 21, 23, TNF-α, IL-10, and TGF-β in the serum was quantified by commercial ELISA kits (Multi Sciences Biotech, Hangzhou, China) according to the instruction of the manufacturer. The BioTek MQX200 microplate reader (BioTek Instruments Inc., Winooski, VT, USA) was used to analyze the optical density of colorimetric reaction at 450 nm. The standard curve was processed according to the optical density and the concentration of the standers using the mELISA software (QINMS, Guilin, China).
3
Cell Migration and Invasion Assay
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Cell migration and invasion were analyzed using 24-well Boyden chambers with an 8-µm pore size polycarbonate membrane. The insert chambers were placed in a 24-well plate, containing 800 µl culture medium with 30% FBS. For the invasion assays, the Matrigel (cat. no. 356234, Corning Inc.) was 1:7 diluted with cold serum-free medium, placed onto the chamber membrane in advance and incubated at 37°C for at least 2 h. In brief, following 24 h of transfection, 6×105 cells (for migration) or 8×105 cells (for invasion) were re-suspended in serum-free culture medium and seeded in the upper chamber and incubated at 37°C for a further 36 h. At the end of the incubation period, the non-migrated or non-invaded cells on the top surface of membrane were removed using cotton swabs, and the migrated or invaded cells on the bottom surface of membrane were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet for 10 min at room temperature. For quantification, the stained migrated or invaded cells were dissolved using 33% acetic acid reagent and the optical densities at 570 nm were determined using an MQX200 microplate reader (BioTek Instruments, Inc.).
4
Caco-2 Cell Cytotoxicity Assay
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The Caco-2 cells concentration was adjusted to 5 × 104 /mL and cultured in a 96-well plate with 200 μL/ hole for 24 h. The experiment was conducted when the confluence of cells reached 80%. BER of different concentrations was diluted in the medium (8 concentration gradients, six replicates for each concentration) 200 μL was added in each hole. The control group (medium containing 10% FBS serum and cells) and the blank group (medium containing 10% FBS serum) were set. They were incubated for 24 h, washed twice with d-hanks solution, and added to 200 μL fresh medium and 20 μL MTT solution (0.5 mg·mL−1), incubating for 4 h. They were washed twice with d-hanks solution, added to with 150 μL DMSO, and oscillated on a plate oscillator for 10 min. The optical density (OD) value of each hole was measured at wavelength 570 nm by MQX200 microplate reader (Bio-Tek, USA). The formula of relative cell survival rate was as follows:
5
Quantifying Cell Viability Using MTT
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Cell viability was assessed using tetrazolium dye (MTT) to quantify the formazan products. After treatment with drugs, MTT reagent (5 mg/mL in PBS) was added to the medium, and the cells were incubated at 37°C for 4 h. The supernatant was subsequently removed, followed by dissolution in DMSO. The absorbance of the solution was measured at a wavelength of 570 nm using an MQX200 microplate reader (Bio-Tek, Winooski, USA). Cell viability is presented as the percentage of that of the control cells.
6
Copper Chloride and Elesclomol Cytotoxicity Assay
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Cells were inoculated into 96-well plates at a rate of 5000 cells/well overnight. Copper chloride solution and elesclomol [32 (link)] were added 1:1 to the 96-well plates at various concentrations of medium. Firstly, we used a larger concentration gradient of copper chloride (0,2.5, 5, 10, 20, 40, 80, 160, 320, 640, (nM)), then, we narrowed the concentration gradient (0, 20, 40, 60, 80, 100, (nM)). After 48 h, CCK-8 (Sevenbio, Beijing, China) reagent was added to each well and then incubated at 37 °C for 2 h. The absorbance at 450 nm was read using a Bio-Tek MQX200 micro-plate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The half maximum inhibitory concentration (half inhibitory concentration) was then calculated.
7
Multimodal Monitoring in ALF Induction
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After ALF induction, all monkeys were monitored every 12 h for the first 48 h and every 24 h for the remainder of the study. During the 6 h treatment, animals were monitored every hour (Figure 1 B ). Vital signs were recorded by cardiogram monitoring. Blood samples were collected for subsequent analyses. Serum biochemistry was assayed using a chemistry analyzer AU5800 series (Beckman Coulter). Ammonia concentrations were quantified by a blood ammonia assay kit (Nanjing Jiancheng, Nanjing, China). S-100 β proteins have emerged as a biomarker of blood-brain barrier (BBB) permeability and neuropathological conditions including encephaledema and increased ICP 13 (link), 14 (link). Thus, elevated S-100 β levels were measured using an ELISA kit (Ruikesi, Chengdu, China). Rhesus IgG and IgM levels were detected using ELISA kits (Ruikesi). All the kits were analyzed with a MQX 200 microplate reader (BioTek Instruments Inc., VT, US). Cytokines were assessed using a Luminex 100 instrument with xPONENT 3.1 software using Monkey Cytokine Magnetic 29-Plex Panel (Invitrogen, CA, US).
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