The analysis of the cell cycle distribution was evaluated by fluorescence imaging analysis of DNA content using a Cytation 1 Cell Imaging Multimode Reader (BioTek, AHSI, Milan, Italy), according to a previous method with minor changes [42 (link)]. To this end, the cells were treated as reported for the γH2AX analysis, then fixed in pure methanol and stained by Hoechst 33,258 (1 µg/mL) dye, which selectively binds DNA into nucleus, leading to a total fluorescence intensity proportionate to the DNA content of cell. Fluorescence images were captured and deconvoluted by Gen5™ Microplate Reader and Imager Software (Version 3.11, BioTek, AHSI, Milan, Italy); moreover, the fluorescence intensity of the Hoechst 33,258-stained nuclei and the relative area were determined. At least 500 cells per images were evaluated and the histograms relating the total fluorescence to the % count were generated using Gen5™ Microplate Reader and Imager Software (Version 3.11, BioTek, AHSI, Milan, Italy), according to the protocol described by the producer [43 ]. The histogram plots were subsequently used to identify G1 and G2 cell cycle phases, based on the prior biological knowledge that the DNA content of G2 cells is doubled with respect to the G1 cells, while the S-phase cells were in the intervening region between the G1 and G2 cells.
Gen5 microplate reader and imager software
Manufactured by Agilent Technologies
Sourced in United States, Italy
The Gen5 Microplate Reader and Imager Software is a versatile data analysis and image capture tool developed by Agilent Technologies. It provides functionality for controlling the operation of microplate readers and imagers, as well as the ability to analyze the collected data.
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Lab products found in correlation
Synergy neo2, by Agilent Technologies (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
High protein binding 96 well elisa plates, by Corning (3 mentions)
Fsl biotin, by Merck Group (3 mentions)
1 steptm ultra tmb elisa substrate solution, by Thermo Fisher Scientific (3 mentions)
Piercetm high sensitivity streptavidin horseradish peroxidase hrp conjugate, by Thermo Fisher Scientific (3 mentions)
Wallac 1420 victor2tm, by Agilent Technologies (3 mentions)
Cytation 5 cell imaging, by Agilent Technologies (3 mentions)
Legend max mouse tslp elisa kit, by BioLegend (2 mentions)
Cytation 1 cell imaging multi mode reader, by Agilent Technologies (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay mts, by Promega (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Elx800 microplate reader, by Agilent Technologies (1 mentions)
96 well round bottom plate, by Corning (1 mentions)
Ready set go, by Thermo Fisher Scientific (1 mentions)
Cytation 3 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
Synergy h1 hybrid multi mode, by Agilent Technologies (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
Multisensitive phosphor screen, by PerkinElmer (1 mentions)
Immobilon, by Merck Group (1 mentions)
28 protocols using gen5 microplate reader and imager software
1
Cell Cycle Analysis by Fluorescence Imaging
2
Quantification of IgA in Ruminant Tissues
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The serum samples were diluted with deionised water to 2000 folds. One hundred milligrams of the mucosa of rumen, duodenum, jejunum and ileum tissue samples were mixed with 1 ml of 0.01 M phosphate buffer solution, pH 7.4 (Sigma, MO, USA), sonicated in an ice-cold water bath for 30 min and stored overnight at −20 °C. Then, thawed mixtures were centrifuged at 5000 × g for 5 minutes in 4 °C. The supernatant was collected and freshly used for the analysis of IgA concentration. The concentration of IgA in serum, rumen, and small intestine tissues (duodenum, jejunum and ileum) were analysed using the Sheep IgA ELISA kit (Life Diagnostics Inc., USA) following manufacturer’s instruction. The absorbance was measured at 450 nm using microplate reader (BioTek™ ELx800™, USA). The concentration of the IgA in samples was determined by substituting the absorbance of samples into the standard curve of standards with known concentration using Gen5 Microplate Reader and Imager Software (BioTek, USA).
3
Quantifying TSLP Levels in Mice
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TSLP level was measured in mice serum using the LEGEND MAX Mouse TSLP ELISA Kit (BioLegend, San Diego, CA) following manufacturer instructions. Optical densities were measured on a Synergy Neo2 (Biotek, Winooski, VT) at 450 nm, and cytokine concentrations were calculated with a five-parameter logistic curve using Gen5 Microplate Reader and Imager Software (Biotek).
4
Serum Lipid Profiling in Veterinary Medicine
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The serum biochemistry was analyzed at the Veterinary Hematology and Clinical Biochemistry Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia. Serum samples were analyzed for cholesterol (TC), triacylglycerol (TAG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and lipase using the Roche/Hitachi 902 clinical chemistry automatic analyzer (Roche, Basel, Switzerland) using appropriate kits. The very-low-density lipoprotein (VLDL) in serum was determined using a Chicken VLDL ELISA kit (FineTest, Wuhan, Hubei, China) according to the protocol provided by the manufacturer. The ELISA kit was based on a double antibody to capture and detect the target protein. The absorbance was determined at 450 nm using an ELx800™ microplate reader (BioTek™, Winooski, Vermont, USA) equipped with Gen5 Microplate Reader and Imager Software (BioTek™, Winooski, VT, USA). The concentration of VLDL was interpolated based on the constructed standard curve of different concentrations of VLDL (μg/ml) against absorbance at 450 nm.
5
Quantifying CAR T-cell Cytokine Secretion
6
NanoGlo Luciferase Assay for T. vaginalis
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The linear dynamic range of the NanoGlo Luciferase Assay System (Promega) in T. vaginalis parasites was determined per the manufacturer’s instructions. Briefly, the NanoGlo reagent was prepared by mixing the NanoGlo substrate in NanoGlo buffer at a ratio of 1:50. Serial dilutions of T. vaginalis-Nluc parasites were prepared (ranging from 0 parasites/100 μL to 106 parasites/100 μL) by diluting parasites in 1x MUT lysis buffer. 100 μL of protein dilutions were added to the wells of a 96-well, round bottom white plate (Corning) in triplicate wells and mixed with 100 μL NanoGlo reagent per well. Luminescence was read using a Synergy H1 Hybrid Multi Mode (BioTek) plate reader at 460 nm. Data was analyzed using Gen5 Microplate Reader and Imager software (BioTek) and Microsoft Excel.
Measuring luminescence of mouse MUT tissue inoculated with Nluc-expressing T. vaginalis parasites was carried out similarly with one minor difference. 100 μL of the processed MUT tissue supernatant was added to the wells of a 96-well, round bottom white plate in triplicate and mixed with 100 μL NanoGlo reagent. The samples were read and analyzed as described above.
Measuring luminescence of mouse MUT tissue inoculated with Nluc-expressing T. vaginalis parasites was carried out similarly with one minor difference. 100 μL of the processed MUT tissue supernatant was added to the wells of a 96-well, round bottom white plate in triplicate and mixed with 100 μL NanoGlo reagent. The samples were read and analyzed as described above.
7
Growth Curves of Klebsiella pneumoniae
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The growth curves of K. pneumoniae were established in LB medium manually, followed by standard protocol with minor modifications. Overnight cultures were diluted to an OD600 of 0.02 and grown in 96-well plates at 37°C and 220 rpm with shaking. The absorbance of the culture solution at OD600 was measured every 0.5 h until it reached the peak and became flat. The OD600 was measured by Gen5 Microplate Reader and Imager Software (BioTek Instruments, https://www.biotek.com/ ). Experiments were performed using three technical replicates and three biological replicates.
8
Evaluating Klebsiella pneumoniae Viscosity
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The viscosity of K. pneumoniae was determined using the string test. Strains that formed strings 5 mm or longer after stretching with the tip of a sterile inoculation loop were considered to have a hypermucoviscous phenotype (Shon et al., 2013 (link)). K. pneumoniae was cultured overnight in LB liquid medium at 37°C and 220 rpm. The cultures were diluted the following day to an OD600 of 1 and centrifuged at 2,350 × g for 5 min, and the OD600 of the supernatant was measured every minute by Gen5 Microplate Reader and Imager Software (BioTek Instruments, https://www.biotek.com/ ). Experiments were performed using three technical replicates and three biological replicates.
9
Protein Expression and Imaging of αVβ3 Integrin
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mMSCs and human umbilical vein endothelial cells (HUVEC, known to express αVβ3) were washed three times in sterile phosphate-buffered saline (PBS). Cell pellets were collected by centrifugation and incubated in a solution of RIPA buffer (Sigma-Aldrich, USA) and Protease Inhibitor (Sigma-Aldrich, USA). The cocktail was sonicated 3 times for a length of 20 seconds with a 10 second incubation on ice. The lysed cells were centrifuged for 15 min and the supernatant (protein) was collected. The concentration of protein was determined using Gen5 Microplate Reader and Imager Software (BioTek Instruments, USA). A total of 20-10 ng of proteins were resolved on 10% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Immobilon, Millipore). Membranes were blocked in Tris-buffered saline (TBS) containing 10% non-fat dry milk and 0.1% Tween 20, prior to incubation with 80 µCi of 64Cu-NOTA-PEG4-cRGD2 targeted to αVβ3 (a well characterized marker of angiogenesis) for 1 hour at room temperature. After washing the membrane with PBS, they were transferred on the multisensitive phosphor screen (Perkin-Elmer, USA) and imaged with Cyclone Plus Storage Phosphor System (Perkin-Elmer, USA).
10
Quantifying PROTAC-Mediated VBC-p38α Interaction
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