The release of lactate dehydrogenase (LDH) was measured in keratinocyte supernatants of both co-cultures and from the LPS assay to assess cell viability. For the LDH assay, a 50-µL aliquot of cell supernatant of each experimental condition was mixed in a 96-well plate with 100 µL of TOX7 reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Absorbance was read on a Multiscan FC microplate reader (Thermo-Fisher Scientific) at 490 and 690 nm. The percentage of LDH release was calculated as described by [23 (link)]. The experiment was performed considering three independent biological replicates according to [10 (link)].
Multiscan fc microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo Scientific Multiscan FC microplate reader is a compact and versatile instrument designed for absorbance-based assays. It features a wide wavelength range, allowing for a variety of colorimetric and fluorometric measurements. The Multiscan FC is capable of reading 6- to 384-well microplates, supporting a wide range of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ldh cytotoxicity colorimetric assay kit 2, by Abcam (3 mentions)
1 step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (2 mentions)
Nunc maxisorp high protein binding capacity 96 well elisa plates, by Thermo Fisher Scientific (2 mentions)
96 well microplate, by Thermo Fisher Scientific (1 mentions)
Formamide, by Fujifilm (1 mentions)
Evans blue, by Fujifilm (1 mentions)
Lactate dehydrogenase activity assay kit, by Merck Group (1 mentions)
Mtt test kit, by Merck Group (1 mentions)
Wst 1 test kit, by Roche (1 mentions)
Ifn gamma duoset elisa, by R&D Systems (1 mentions)
Spectramax microplate reader, by Molecular Devices (1 mentions)
Luciferin, by Promega (1 mentions)
Calcusyn software, by Biosoft (1 mentions)
Thermostat, by Thermo Fisher Scientific (1 mentions)
D luciferin, by Thermo Fisher Scientific (1 mentions)
12 well culture plate, by Corning (1 mentions)
Mtt cell growth assay kit, by Merck Group (1 mentions)
Nsc 74859, by Bio-Techne (1 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
U0126, by Bio-Techne (1 mentions)
31 protocols using multiscan fc microplate reader
1
Measurement of Cell Viability via LDH Release
2
Antioxidant Activity Determination Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antioxidant activity was determined using the free radicals DPPH• and ABTS•+ spectrophotometric methods adapted to a microscale in accordance with previously described methods by [40 (link)]. The measurements were performed using 96-well microplates (Nunc, Roskilde, Denmark) and measured by a Multiscan FC microplate reader (Thermo-Fisher Scientific, Inc., Waltham, MA, USA). A total of three technical replicates of each sample were assessed. The results were expressed as mM Trolox g−1 of cowpea tissues dry weight (mM Trolox g−1 dw).
3
Measuring Rat Plasma Corticosterone
4
Ganglioside-Binding Assay for Antibody Fragments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nunc MaxiSorp high protein-binding capacity 96-well ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with gangliosides GD2, GM2, GD1b, and GD3 at a concentration of 0.25 μg in 100 μL of 96% ethanol per well. GD2, GD1b, and GD3 were purchased from Sigma-Aldrich, and GM2 was obtained according to our previous work [35 (link)]. Following air drying, plate wells were blocked with 100 μL 2% BSA in PBS supplemented with 0.1% Tween-20 (PBS-T) per well for 2 h at RT. ScFv fragments, minibodies, or FDCs (100 μL solution in PBS-T per well) were added in triplicates at different concentrations, and incubation was carried out for 1.5 h. After washing with PBS-T, anti-FLAG HRP-labeled antibodies were added to the wells with scFv fragments, and anti-human Fc-specific HRP-labeled antibodies were added to the wells with minibodies (both 1:6000; Santa Cruz Biotechnology, CA, USA). After 40 min incubation and further washing, 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Waltham, MA, USA) was added, and the color reaction OD was measured at 450 nm by Multiscan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Percent of cross-reactivity was calculated as the ratio of TMB color reaction OD450 in GM2-, GD1b-, or GD3-coated wells to OD450 in GD2-coated wells.
5
Quantifying Blood-Brain Barrier Breakdown in Malaria
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
It is known that breakdown of the blood–brain barrier is an indicator of ECM. When P. berghei-infected mice develop ECM, Evans blue is injected i.v. and the brain is stained as a result of extravasation of the dye [20 (link)]. Mice were injected i.v. with 0.2 mL of 1% (w/v) Evans blue (Wako, Osaka, Japan) on days 7 and 14 post-infection. Mice were euthanized and brains perfused with PBS 1 h later. After the brains were removed and photographed they were weighed and placed in 2 mL formamide (Wako, Osaka, Japan) at 37 °C for 48 h to extract the Evans blue dye. Absorbance was measured at λ = 620 nm with a Multiscan FC microplate reader (Thermo Fisher Scientific Inc., Waltham, USA). The Evans blue concentration was calculated from a standard curve and is expressed as µg of Evans blue per g of brain.
6
Evaluating Lactate Dehydrogenase in Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following the manufacturer’s instructions, the LDH activity intracellularly and in the cell culture supernatant was assessed using a Lactate Dehydrogenase activity assay kit (Sigma-Aldrich). After 3 h, 24 h, and 48 h of co-culture, the cell culture supernatant was collected and preserved at −80 °C for subsequent LDH activity measurements. Adherent cells were trypsinized and washed with Phosphate-Buffered Saline. The cellular pellets were resuspended in 350 µL of cold LDH Assay Buffer. Subsequently, this suspension was centrifuged at 10,000× g for 15 min at 4 °C to eliminate cellular debris, including fungal cells in the co-culture, and the resulting soluble fraction was stored at −80 °C for later LDH activity assessments.
LDH activity was ascertained based on NADH oxidation, and absorbance readings were recorded at 450 nm using a Multiscan FC microplate reader (Thermo Fisher Scientific). Uninfected HaCaT cells were used as controls. For quantifying LDH activity in the cell supernatant, we used RPMI-1640 containing 5% fetal bovine serum as a negative control. Simultaneously, LDH Assay Buffer was utilized as a negative control for intracellular LDH activity.
Three independent biological replicates were performed for each experimental condition.
LDH activity was ascertained based on NADH oxidation, and absorbance readings were recorded at 450 nm using a Multiscan FC microplate reader (Thermo Fisher Scientific). Uninfected HaCaT cells were used as controls. For quantifying LDH activity in the cell supernatant, we used RPMI-1640 containing 5% fetal bovine serum as a negative control. Simultaneously, LDH Assay Buffer was utilized as a negative control for intracellular LDH activity.
Three independent biological replicates were performed for each experimental condition.
7
MTT Assay for Cell Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was assessed with MTT-test kit (Sigma). Briefly, SH-SY5Y cells were seeded in 96-well plates and cultured for 24 h at 37 °C. Then, the cells were treated with isoAβ42 or Aβ42 for 48 h followed by incubation with MTT reagent for 4 h at 37 °C. The absorbance of samples was measured in a multiscan FC microplate reader (Thermo Fisher Scientific) at 570 nm. Viability of untreated cells was taken as 100%.
8
Measuring Cell Viability with WST-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was assessed with a WST-1 test kit (Roche Diagnostics), which is based on the cleavage of water-soluble tetrazolium salt by mitochondrial dehydrogenases in live cells. SiHa cells were seeded in 96-well plates and cultured for 24 h at 37°C. Then, the cells were treated with binase and/or IFNα2b for 48-72 h followed by incubation with WST-1 reagent for 60 min at 37°C. The absorbance of samples was measured in a multiscan FC microplate reader (Thermo Fisher Scientific) at 450 nm. A mixture of cell-free medium with the WST-1 reagent was used as a background control. The activity of mitochondrial dehydrogenases was calculated as the difference in absorbance between each sample and the background control. Respiratory activity of untreated cells was taken as 100%. The experiment was performed in triplicate and reported as mean± SD.
9
Quantifying T Cell Cytotoxicity and IFNγ Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the assessment of IFNγ production, T cells were cocultured with target cell lines at cell counts and effector-to-target (E:T) ratio indicated in the figure legend in 96-well round-bottom plate at a final volume of 200 μl per well. After overnight coculture, the supernatant was harvested and the levels of IFNγ were assessed using enzyme-linked immunosorbent assay (ELISA) (kits: IFN-gamma DuoSet ELISA DY285 for human IFNγ and DY485 for mouse IFNγ, R&D Systems; Multiscan FC Microplate Reader, Thermo Fisher Scientific). For in vitro cytotoxicity measurement, target cell lines that are naturally HLA-A*02:01+ and CD20+ were virally transduced to express firefly luciferase. T cells were cocultured with firefly luciferase–expressing target cell lines at E:T ratios indicated in the figure legend for 6 hours. Upon completion of coculture, luciferin (Promega) was added to the well and the level of luminescence was measured with a luminometer (SpectraMax Microplate Reader, Molecular Devices LLC). For HLA-blocking experiments, target cells were incubated with 50 μg/ml of HLA-blocking antibodies or an isotype control for 3 hours in 37°C, and then T cells were added to each condition as indicated in the figure legend. HLA-blocking antibody clones and sources are listed in table S7.
10
Antioxidant Capacity Evaluation of Beans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The free radical scavenging (DPPH and ABTS) assays and ferric reducing antioxidant power (FRAP) assay were performed according to the methods described by Espín et al. [30 (link)], with slight modifications. The measurements were performed at microscale using 96-well microplates and measured by a Multiscan FC microplate reader (Thermo-Fisher Scientific, Inc., Waltham, MA, USA). In DPPH and ABTS assays, 12 µL of bean extract were added to 188 µL of working solution to complete the volume reaction of 200 µL. The absorbance was measured at room temperature after 30 min of reaction at 520 nm for DPPH and at 734 nm for ABTS. In the FRAP assay, the total reaction volume was 300 µL, corresponding to 12 µL of bean extract and 288 µL of working FRAP solution. The mixtures were incubated for 30 min at 37 °C in the dark and read at 593 nm. The results of all procedures were measured as µM of trolox equivalents per gram of dry weight (µM TE/g dw).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!