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Filtermax f3 f5 multi mode microplate reader

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The FilterMax F3 & F5 Multi-Mode Microplate Readers are compact, high-performance instruments designed for a variety of microplate-based assays. They provide accurate and reliable absorbance, fluorescence, and luminescence measurements in a single platform.

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Lab products found in correlation

Human homocysteine hcy elisa kit, by MyBioSource (2 mentions) Glucose uptake cell based assay kit, by Cayman Chemical (2 mentions) Maxisorp, by Thermo Fisher Scientific (2 mentions) A8592, by Merck Group (2 mentions) Anti m13 mab, by GE Healthcare (2 mentions) Adenosylhomocysteinase activity fluorometric assay kit, by Abcam (1 mentions)

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9 protocols using filtermax f3 f5 multi mode microplate reader

1

Quantifying SAHH Activity in Cells

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The experiments were performed in a 96-well scale using the Human Homocysteine (Hcy) ELISA Kit (Mybiosource, San Diego, CA, USA; MBS260128) that allows quantitative measurement of homocysteine concentration in cell extracts, according to the manufacturer's instructions. The concentration of homocysteine in cell extracts was used as a readout for in vivo SAHH activity,8 (link) as SAHH hydrolyzes SAH to homocysteine and adenosine. Briefly, ARK2 cells were washed with cold phosphate-buffered saline and lysed on plate in 200 μl of lysis buffer (40 mm hexadecyltrimethylammonium bromide, 75 mm Tris-HCl, pH 8.0, 1m NaCl, 15 mm EDTA). The lysate was cleared of insoluble materials by centrifugation at 15 000 g at 4 °C for 15 min. Immediately following the centrifugation, 100 μl of the supernatant was collected and used for SAHH activity measurement. The absorbance of the samples was determined using a FilterMax F3&F5 Multi-Mode Microplate Reader (Molecular Devices).
Zhong T., Men Y., Lu L., Geng T., Zhou J., Mitsuhashi A., Shozu M., Maihle N.J., Carmichael G.G., Taylor H.S, & Huang Y. (2016). Metformin alters DNA methylation genome-wide via the H19/SAHH axis. Oncogene, 36(17), 2345-2354.
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2

Glucose Uptake Assay in Myotubes

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These were performed in a 96-well plate scale. Glucose uptake assays were carried out using the Glucose Uptake Cell-Based Assay Kit (Cayman Chemical, catalog number 600470) according to the manufacturer's instructions with minor modifications. Briefly, 48 h after siRNA transfection, myotubes were incubated in 200 μl of glucose-free DMEM (Gibco, catalog number 11966–025) in the presence or absence of 100 nM of insulin for 2 h. The medium was then removed and replaced with 100 μl of new glucose-free DMEM containing fluorescent 2-NBDG at a final concentration of 150 μg/ml. Incubation was carried out in dark for an additional 10 min in the tissue culture incubator. The medium was then removed and the cells washed once with 200 μl of ice-cold phosphate buffered saline (PBS). After adding 100 μl of new ice-cold PBS to the cells, fluorescent intensity was immediately determined using the fluorescent plate reader (FilterMax F3&F5 Multi-Mode Microplate Reader, Molecular Devices).
Gao Y., Wu F., Zhou J., Yan L., Jurczak M.J., Lee H.Y., Yang L., Mueller M., Zhou X.B., Dandolo L., Szendroedi J., Roden M., Flannery C., Taylor H., Carmichael G.G., Shulman G.I, & Huang Y. (2014). The H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells. Nucleic Acids Research, 42(22), 13799-13811.
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3

Quantifying SAHH Activity via Homocysteine ELISA

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The experiments were performed in a 96-well scale using the Mouse Homocysteine (Hcy) ELISA Kit (Mybiosource, MBS260152) that allows quantitative measurement of homocysteine concentration in cell extracts, according to the manufacturer's instructions. The concentration of homocysteine in cell extracts was used as a readout for in vivo SAHH activity37 (link), as SAHH hydrolyses SAH to homocysteine and adenosine. Briefly, myotubes were washed with cold PBS and lysed on plate in 200 μl of lysis buffer (40 mM hexadecyltrimethylammonium bromide, 75 mM Tris-HCl, pH 8.0, 1 M NaCl, 15 mM EDTA). The lysate was cleared of insoluble materials by centrifugation at 15,000g at 4 °C for 15 min. Immediately following the centrifugation, 100 μl of the supernatant was collected and used for SAHH activity measurement. The absorbance of the samples was determined using a FilterMax F3&F5 Multi-Mode Microplate Reader (Molecular Devices). A serial dilution of mouse Hcy was included in each assay to obtain a standard curve. The assay was performed in triplicate. The concentrations of DNA and RNA extracted from parallel wells were used as loading controls for SAHH activity normalization.
Zhou J., Yang L., Zhong T., Mueller M., Men Y., Zhang N., Xie J., Giang K., Chung H., Sun X., Lu L., Carmichael G.G., Taylor H.S, & Huang Y. (2015). H19 lncRNA alters DNA methylation genome wide by regulating S-adenosylhomocysteine hydrolase. Nature Communications, 6, 10221.
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4

Lipid Peroxidation Quantification by TBARS

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Lipid peroxidation in the cells was estimated using the thiobarbituric acid-reactive substance (TBARS) assay [26 (link)]. 0.25 mL 15% (W/V) trichloroacetic acid and 7 μL of 500 mM butylated hydroxyanisole (BHA) were added to the cell lysate. The mixture were then centrifuged at 1000 g for 5 min. The supernatant was taken and 0.5 ml 0.375% (w/v) thiobarbituric acid was added. The mixture was then boiled for 10 min. After the mixture was cooled, TBARS was measured at 532 nm using a microplate reader (FilterMax F3/F5 Multi-Mode Microplate Reader, Molecular Devices).
Han F., Li S., Yang Y, & Bai Z. (2021). Interleukin-6 promotes ferroptosis in bronchial epithelial cells by inducing reactive oxygen species-dependent lipid peroxidation and disrupting iron homeostasis. Bioengineered, 12(1), 5279-5288.
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5

Fluorescent Glucose Uptake Assay in Myotubes

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The glucose uptake assay was performed on in vitro differentiated mouse and human myotubes in a 96-well plate using the Glucose Uptake Cell-Based Assay Kit (Cayman Chemical, MI, USA, catalogue no. 600470) according to the manufacturer’s instructions with minor modifications. On the day of the assay, culture media were replaced with 200 μl of glucose-free DMEM (Gibco, catalogue number 11966-025) and incubation was carried out for 2 h. Then, the medium was replaced with 100 μl of new glucose-free DMEM in the presence or absence of 100 nmol/l of insulin for 15–20 min. Subsequently, 100 μl of new glucose-free DMEM containing fluorescent 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose at a final concentration of 150 μg/ml was added. Incubation was carried out in the dark for an additional 15 min in a tissue culture incubator. The medium was then removed and the myotubes were washed once with 200 μl of ice-cold PBS. After adding 100 μl of new ice-cold PBS to the myotubes, fluorescent intensity was immediately determined using the fluorescent plate reader (FilterMax F3&F5 Multi-Mode Microplate Reader; Molecular Devices, CA, USA). Results are presented with NT siRNA-transfected myotubes without insulin stimulation arbitrarily set as 1.
Liu B., Xie D., Huang X., Jin S., Dai Y., Sun X., Li D., Bennett A.M., Diano S, & Huang Y. (2024). Skeletal muscle TET3 promotes insulin resistance through destabilisation of PGC-1α. Diabetologia, 67(4), 724-737.
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6

Measuring H19 RNA Inhibition of rSAHH

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These experiments were designed to test whether binding of H19 RNA fragments to rSAHH would inhibit rSAHH enzymatic activity in vitro. The experiments were performed using the Adenosylhomocysteinase Activity Fluorometric Assay Kit (Biovision, K807-100) according to the manufacturer's instructions. This kit allows quantitative detection of adenosine concentration as a readout for rSAHH's ability to hydrolyse SAH in vitro. Briefly, 2 mg of rSAHH was pre-incubated with unlabelled RNA fragments (preheated at 90 °C for 3 min and then chilled on ice before use) in AHCY assay buffer in a total volume of 20 μl at room temperature for 20 min. At the end of incubation, an additional 30 ml of AHCY assay buffer was added, followed by addition of 50 μl of reagents containing SAH. The resulting 100 μl reaction mixture was immediately subjected to absorbance reading using a kinetic mode in a FilterMax F3&F5 Multi-Mode Microplate Reader (Molecular Devices). In the final 100-μl reaction mixture, the concentration of rSAHH was 0.4 mM, and those of the RNA fragments were 0, 0.2 and 1 nM. Relative rSAHH activities are presented with those in the absence of the RNA fragments arbitrarily set as 1.
Zhou J., Yang L., Zhong T., Mueller M., Men Y., Zhang N., Xie J., Giang K., Chung H., Sun X., Lu L., Carmichael G.G., Taylor H.S, & Huang Y. (2015). H19 lncRNA alters DNA methylation genome wide by regulating S-adenosylhomocysteine hydrolase. Nature Communications, 6, 10221.
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7

Screening of Anti-CCR2 scFv Antibodies

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Nunc MaxiSorp 96-well plates were coated with NeutrAvidin at 5 μg/mL and incubated overnight at 4 °C. The plate was thoroughly washed with 0.1 % PBS-Tween (PBST), and 5 μg/mL biotinylated human ECL2 CCR2 peptide (TKCQKEDSVYVCGPYFPRGWNNFHTIMR) was added and incubated for 1 h at room temperature. The wells were washed with 0.1 % PBST and then blocked with casein blocking buffer for 1 h at room temperature. The wells were washed with 0.1 % PBST followed by incubation of serially diluted scFv for 1 h at room temperature before being washed five times with 0.1 % PBST. Washes were followed by the addition of 100 μL of the respective horseradish peroxidase (HRP)-linked monoclonal antibody (mAb) for 1 h at room temperature with rocking. An anti-M13 mAb was used to detect phage clones (#27–9421-01, GE Healthcare, Chicago, IL) and an anti-FLAG mAb was used to detect scFv protein (#A8592, Sigma Aldrich, St. Louis, MO). The wells were washed with 0.1 % PBST followed by the addition of TMB-ELISA substrate. After 10 min incubation, 2 M H2SO4 was added to stop the reaction. The absorbance was measured at 450 nm with a FilterMax F3 & F5 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA). A 1:1 binding ratio Hill model was used to fit the binding interaction data.
, & Collins K.D. (1995). Sticky ions in biological systems.. Proceedings of the National Academy of Sciences of the United States of America, 92(12), 5553-5557.
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8

Quantitative Measurement of SAHH Activity

Cited 1 time
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The experiments were performed in a 96-well scale using the Human Homocysteine (Hcy) ELISA Kit (Mybiosource, San Diego, CA, USA; MBS260128) that allows quantitative measurement of homocysteine concentration in cell extracts, according to the manufacturer's instructions. The concentration of homocysteine in cell extracts was used as a readout for in vivo SAHH activity,8 (link) as SAHH hydrolyzes SAH to homocysteine and adenosine. Briefly, ARK2 cells were washed with cold phosphate-buffered saline and lysed on plate in 200 μl of lysis buffer (40 mm hexadecyltrimethylammonium bromide, 75 mm Tris-HCl, pH 8.0, 1m NaCl, 15 mm EDTA). The lysate was cleared of insoluble materials by centrifugation at 15 000 g at 4 °C for 15 min. Immediately following the centrifugation, 100 μl of the supernatant was collected and used for SAHH activity measurement. The absorbance of the samples was determined using a FilterMax F3&F5 Multi-Mode Microplate Reader (Molecular Devices).
Zhong T., Men Y., Lu L., Geng T., Zhou J., Mitsuhashi A., Shozu M., Maihle N.J., Carmichael G.G., Taylor H.S, & Huang Y. (2016). Metformin alters DNA methylation genome-wide via the H19/SAHH axis. Oncogene, 36(17), 2345-2354.
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9

Peptide-Binding Assay for CCR2 scFv

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Nunc MaxiSorp 96-well plates were coated with NeutrAvidin at 5 µg/mL and incubated overnight at 4°C. The plate was thoroughly washed with 0.1% PBS-Tween (PBST), and 5 µg/mL biotinylated N-terminal domain CCR2 peptide (MEDNNMLPQFIHGILSTSHSLFTRSIQELDEGATTPYDYDDGEPC; ABclonal, Woburn, MA) added and incubated for 1 h at room temperature. The wells were washed with 0.1% PBST and then blocked with casein blocking buffer for 1 h at room temperature. The wells were washed with 0.1% PBST followed by incubation of serially diluted scFv for 1 h at room temperature before being washed five times with 0.1% PBST. Washes were followed by the addition of 100 uL of the respective horse-radish peroxidase (HRP)-linked monoclonal antibody (mAb) for 1 h at room temperature with rocking. An anti-M13 mAb was used to detect phages (#27–9421-01, GE healthcare, Chicago, IL) and an anti-flag mAb was used to detect scFv (#A8592, Sigma Aldrich, St. Louis, MO). The wells were washed with 0.1% PBST followed by the addition of TMB-ELISA substrate. After 10 min incubation, 2 M H2SO4 was added to stop the reaction. The absorbance was measured at 450 nm with a FilterMax F3 & F5 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA). A 1:1 binding ratio Hill model was used to fit the binding interaction data.
Deci M.B., Ferguson S.W., Scatigno S.L, & Nguyen J. (2018). Modulating macrophage polarization through CCR2 inhibition and multivalent engagement. Molecular pharmaceutics, 15(7), 2721-2731.
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