Metabolic activity of the bacteria or yeast was determined using alamarBlue® assays designed to measure quantitatively the proliferation of various human and animal cell lines, bacteria and fungi according to the manufacturer's instructions (Thermo Fisher Scienti c DAL1025). Brie y, the assay measures the natural reducing power of living cells to convert resazurin, a cell permeable compound that is blue in colour and virtually non-uorescent, into the bright red-uorescent resoru n. The amount of uorescence produced is proportional to the number of living cells. 10µl of alamarBlue® was added into 100µl of cell suspension and incubated for 2 hrs. The uorescence of the alamarBlue® assay plate was read with a Tecan In nite M200 reader using an excitation between 530-560 nm and an emission at 590 nm. Normalized metabolic activity was calculated as a percentage of the alarmaBlue Relative Light Units (RLU) measured in the Tecan reader for the control, no-treated sample set as 100% compared to the RLU measured for the treated sample(s).
In nite m200
Manufactured by Tecan
Sourced in Austria, China
The Infinite M200 is a multi-mode microplate reader designed for a range of applications in life science research and drug discovery. It is capable of performing absorbance, fluorescence, and luminescence measurements with high sensitivity and precision.
Automatically generated - may contain errors
6 protocols using in nite m200
1
Quantifying Metabolic Activity in Cells
2
Cell Viability Assay for PCBP1 Overexpression
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Cell viability was investigated using the methyl tetrazolium salt (MTS) assay. Cells were plated into 96-well plates (Promega, Beijing, China) and incubated 24 h. Then used transfection reagent to transfect pEGFP-N1 or pEGFP-N1-PCBP1 and cultured 24 h and 48 h to detect viability. 20 µl / well of MTS solution was added to each 100 µl of DMEM medium, and incubated for 60 min at a 37℃ constant temperature incubator. Then the absorbance was detected with multifunction microplate reader (Tecan In nite M200, Swiss) at 490 nm. The survival rate of cells in each well was shown as a percentage of control.
Quantitative RT-PCR analysis and agarose gel electrophoresis Total RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2 -ΔΔCT ) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels.
Quantitative RT-PCR analysis and agarose gel electrophoresis Total RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2 -ΔΔCT ) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels.
3
Soil Enzyme Activity Assay Protocol
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The activity of four soil enzymes involved in SOM decomposition and soil nutrient cycling were measured using uorescence-based enzyme activity assay (Table 3). Rhizosphere soil samples from each pot were analyzed using the microplate enzyme assay using uorescence-based MUB (4-methylumbelliferone) and MUC (7-amino-4-methylcoumarin) substrate protocol (Bell et al. 2013 ). Brie y, the day after the plants were harvested, 1.1-1.3 g of soil was weighed from the rhizosphere soil sample. The soil was blended to homogenize sample with a 50mM sodium acetate buffer solution, that had been adjusted to the average soil pH of 7.5 to make a soil slurry. Soil slurry was pipetted into black, 96-well microplates with compound-speci c uorescing substrates. Samples were analyzed using a Tecan In nite M200 plate reader (Tecan Austria GmbH, Salzburg, Austria).
4
Serum lipid profiling in mice
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Sera were collected from the mice. Serum lipids, including triglycerides, total cholesterol, LDLs and HDLs, were determined based on commercial kits (Jiancheng, Nanjing, China) using a microplate reader (Tecan In nite M200).
5
Measuring IL-6 Levels in Mouse Plasma
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Plasma was collected from the mice. Then, the levels of IL-6 were assessed with ELISA kits (Lianke Biotech, Hangzhou, China) according to the manufacturer's instructions. The absorbance was measured using a microplate reader (Tecan In nite M200).
6
Cell Viability Assay for PCBP1 Overexpression
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Cell viability was investigated using the methyl tetrazolium salt (MTS) assay. Cells were plated into 96-well plates (Promega, Beijing, China) and incubated 24 h. Then used transfection reagent to transfect pEGFP-N1 or pEGFP-N1-PCBP1 and cultured 24 h and 48 h to detect viability. 20 µl / well of MTS solution was added to each 100 µl of DMEM medium, and incubated for 60 min at a 37℃ constant temperature incubator. Then the absorbance was detected with multifunction microplate reader (Tecan In nite M200, Swiss) at 490 nm. The survival rate of cells in each well was shown as a percentage of control.
Quantitative RT-PCR analysis and agarose gel electrophoresis Total RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2 -ΔΔCT ) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels.
Quantitative RT-PCR analysis and agarose gel electrophoresis Total RNA was extracted from cells with TRIzol reagent (Takara Biotech Co., Ltd.) and the complementary DNA (cDNA) was synthesized by using Transcriptor First Strand cDNA Synthesis System kit, real-time PCR analysis of PCBP1 and the reference gene β-Actin was treated by using a SYBR Green reaction kit (TIANGEN, China) in real-time PCR instrument (Thermo, USA), according to instruction. All experiments were carried out in triplicate and analyzed using the comparative threshold cycle (2 -ΔΔCT ) method. Products were run in 1% agarose gel and the band intensity was scanned. The results were normalized by β-Actin levels.
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