The T cell‐mediated cytotoxicity assays were performed by the Calcein‐AM releasing method as previously described.24 Briefly, total 1 × 106 cells/ml of tumor cells were prestained with the dye Calcein‐AM at the concentration of 10 μmol/L at 37°C for 30 min. After washing, prestained tumor cells were treated with the different T cells in the 96‐well plates with different E:T ratios from 2.5:1 to 20:1. After 4 h, the supernatants were harvested. Mean fluorescence intensity was determibed at 495/515 nm using the PerkinElmer Multimode Reader. The killing percentages in the sample wells were calculated according to the formula:
Multimode reader
Manufactured by PerkinElmer
Sourced in United States
The Multimode reader is a versatile laboratory instrument designed to perform a variety of detection methods, including absorbance, fluorescence, and luminescence. It is capable of reading different types of microplates and can be used for various applications in the field of life sciences and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Cck 8 reagent, by Dojindo Laboratories (3 mentions)
Tmb one substrate solution, by Promega (2 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
24 well cell culture dishes, by Sangon (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
96 well plate, by Corning (1 mentions)
D300 digital dispenser, by Hewlett-Packard (1 mentions)
Calcein am, by Merck Group (1 mentions)
Rhodamine b, by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Mtt solution, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Pierce bca kit, by Thermo Fisher Scientific (1 mentions)
96 well elisa plate, by Corning (1 mentions)
Hrp conjugated goat anti human igg, by Southern Biotech (1 mentions)
28 protocols using multimode reader
1
Cytotoxicity Assay for T Cell-Mediated Killing
2
Cell Viability and Proliferation Assays
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Cell Counting Kit-8 (CCK-8) assay: Cell viability was assessed indirectly by CCK-8 assay. SGC-7901 cells were seeded in a 96-well plate at a density of 3000 cells/well. After 24 h, cells were transfected with the indicated miRNAs or siRNAs. Forty-eight hours later, cells were incubated with 100 µL of fresh medium containing 10% CCK-8 reagent (DoJinDo Laboratories, Japan) for 1h at 37℃. Then, the absorbance values of the wells at 450 nm were detected using an automatic spectrometer (Multimode Reader, PerkinElmer, USA). This procEdUre was repeated at 1, 3, 4, 5, 6, and seven days after cell seeding. EdU (5-ethynyl-2'-deoxyuridine) Cell Proliferation Assay: The cells were seeded in 96-well plates at 5×103 cells/well. Forty-eight hours after transfection, EdU (50 μM; Thermo Fisher Scientific, USA) labeling media was added to the plates, and further incubated for two hours at 37℃ in 5% CO2. After being treated with 4% paraformaldehyde and 0.5% Triton X-100, the cells were stained with the anti-EdU working solution. DAPI stained the cell nuclei. Count the numbers both of EdU-positive and negative cell nuclei in five randomly areas and calculated the percentage of EdU-positive cells.
3
Intestinal Permeability Measurement in Colitic Mice
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The intestinal permeability of mice with DSS-induced colitis was measured as previously described (21 (link)). A total of 44 mg/100 g body weight FITC-labeled 4-kDa dextran (FD-4; MW, 4,000 Da; Sigma-Aldrich; Merck KGaA) was administrated to each mouse via gavage following fasting for 4 h. Following FD-4 administration for 5 h, 400 µl blood was collected and centrifuged at 2,500 x g for 10 min at 4˚C to isolate serum. The serum fluorescence intensity was determined using a multimode reader (PerkinElmer, Inc.) at excitation wavelength of 485 nm and emission wavelength of 520 nm. Subsequently, a standard curve was generated using serial dilutions of FD-4.
4
Ajwain Oil Enhances Glucose Uptake
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Antidiabetic activity of ajwain oil was assessed in differentiated L6 myotubes using fluorescent-tagged 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (6-NBDG). L6 myotubes (10,000 cells/well) were seeded in 96-well plates and allowed to confluence around 80%. Then, the cells were differentiated using 2% FBS and different concentrations of the oil (0.25–4 µL/mL) was added. At the end of treatment, 10 µM of insulin was added to stimulate glucose uptake and incubated for 15min. A total of 20 µg/200mL of 6-NBDG was added and incubated for 10min at dark. Glucose uptake (in percentage) was measured using Multimode reader (PerkinElmer) with an excitation/emission filter of 466/540nm.[14 (link)]
5
Isolation and Regulation of Goose Hepatocytes
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Goose hepatocytes were isolated from 1–2 week-old Landes goose following the perfusion method41 (link). Cells were cultured in RPMI1640 medium containing 10% FBS (Gibco, USA) and incubated at 5% CO2, and 37 °C on 24-well cell culture dishes (Sangon, China) at a density of 3 × 105/cm2. Medium was changed every 24 h. Cells were distributed into 4 groups after 24 h growth: blank control group (BC), negative control group (NC), aan-miR-203a mimic group and aan-miR-125b-5p mimic group. Transfections were performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s introductions. Cells were collected after 48 h of tranfection to detect the expression of ACSL1 and ELOVL6. The intracellular lipid contents of hepatocytes were monitored using multimode reader (Perkinelmer, Singapore) after Nile Red staining.
6
SARS-CoV-2 Receptor Binding Domain Protein Evaluation
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96-well plate (Corning, NY, USA) was coated with 2 µg/ml of plant-produced F-native protein, plant-produced SC-TM protein, and plant-produced SARS-CoV-2 Receptor binding domain protein [36 (link)] (as a negative control) in phosphate buffer (20 mM, pH 7.4), 25 µl per well, overnight at 4 °C. After washing three times with PBS-T, the plate was blocked with 200 µL of 5 % skim milk in PBS at 37 °C for 1 h. Then, the plate was washed three times with PBS-T and incubated with serially diluted plant-produced Motavizumab and incubated at 37 °C for another 2 h. After sample incubation, the solution was removed and washed three times with PBS-T. After washing, the plate was incubated with 1:2500 HRP-conjugated goat anti-human IgG kappa light chain (Southern Biotech, Birmingham, AL, USA), and incubation was continued at 37 °C for another hour. After secondary antibody incubation, the wells were emptied and washed. The TMB one substrate solution (Promega, Madison, WI, USA) was added and incubated for 10 min, the reaction was stopped by adding 1 M sulfuric acid and the absorbance was measured using a multimode reader (Perkin Elmer, MA, USA) at 450 nm.
7
Cytotoxicity assessment of plant extract
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Cytotoxicity of the test extract was assessed by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.[13 (link)] The plant extract was prepared in DMSO for the cytotoxicity study. Cells were plated in 48-well plate at a concentration of 5 × 104 cells/well. After 24 h of incubation, it was washed with 200 ml of 1X phosphate buffered saline (PBS; pH 7.4) and starved by incubation in serum-free medium for an hour at 37°C in CO2 incubator. After starvation, cells were treated with different concentrations (1–1000 μg/mL) of the test extract for 24 h. At the end of the treatment, media from control and extract-treated cells were discarded, and 50 μl of MTT containing PBS (5 μg/mL) was added to each well. Cells were then incubated for 4 h at 37°C in CO2 incubator. The purple formazan crystals formed were then dissolved by adding 150 ml of DMSO and mixed effectively by pipetting up and down. Spectrophotometrical absorbance of the purple blue formazan dye was measured using multimode reader (Perkin Elmer) at 570 nm. Optical density of each sample was compared with control optical density and graphs were plotted.
8
Antidiabetic Potential of Extract in L6 Myotubes
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Antidiabetic activity of the test extract was assessed in differentiated L6 myotubes using fluorescent-tagged 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol- 4-yl) amino)-2-deoxyglucose (6-NBDG). L6 myotubes (10,000 cells/well) were seeded in 96-well plates and allow to confluence around 80%. Then, cells were differentiated using 2% FBS, and different concentrations of the extract dissolved in DMSO (1–100 μg/mL) were added. At the end of treatment, 10 μM of insulin was added to stimulate glucose uptake and incubated for 15 min. About 20 μg/200 mL of 6-NBDG was added and incubated for 10 min at dark. Glucose uptake (in %) was measured using multimode reader (Perkin Elmer) with an excitation/emission filter 466/540 nm.
9
Measuring p38α Kinase Inhibition
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A titration of TAB-1 (Ac-RVYPVSVPYSSAQSTSKTSVTLSLVMPSQ-NH2) or MKK3b (Ac-GKSKRKKDLRISCMSKP-NH2) peptide was dispensed from a concentrated solution in DMSO using a Hewlett Packard D300 digital dispenser directly into a 384-well low-volume assay plate. The DMSO concentration was normalized to 1% of the final assay volume. To this was added a mixture of 2 nM fluorescein-labeled MKK3b peptide (Ac-GKSKRKKDLRISCMSKPK[ε-fluorescein]-NH2) and 10 μM p38α in a buffer of 50 mM HEPES pH 7.4, 10 mM MgCl2, 1 mM Chaps, and 1 mM DTT. The final well volume was 10 μl. After incubation at ambient temperature for 20 minutes, fluorescence polarization was measured on a Perkin Elmer multimode reader (480 nm excitation filter, 535 nm emission filters, 505 nm mirror). Data were normalized between averaged control wells of DMSO only (0% displacement) and ligand only (100% displacement). Duplicate titrations were averaged and fitted to a 4-parameter IC50 function.
10
Cytotoxicity Evaluation of Ajwain Oil
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Cytotoxicity of the ajwain oil was assessed by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.[13 (link)] Cells were plated in 48-well plate at a concentration of 5×104 cells/well. After 24h of incubation, it was washed with 200 µl of 1× phosphate-buffered saline (PBS; pH, 7.4) and starved by incubation in serum-free medium for an hour at 37oC in CO2 incubator. After starvation, cells were treated with different concentrations (0.03–4 µL/mL) of the oil for 24h. At the end of the treatment, media from control and oil-treated cells were discarded and 50 µl of MTT-containing PBS (5mg/mL) was added to each well. Cells were then incubated for 4h at 37oC in CO2 incubator. The purple formazan crystals formed were then dissolved by adding 150 µl of dimethyl sulfoxide and mixed effectively by pipetting up and down. Spectrophotometrical absorbance of the purple blue formazan dye was measured using Multimode reader (Perkin Elmer, USA) at 570nm. Optical density of each sample was compared with control optical density and graphs were plotted.
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