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Fl600 microplate reader

Manufactured by Agilent Technologies
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The FL600 microplate reader is a compact and versatile fluorescence detection instrument designed for a wide range of life science and analytical applications. It provides accurate and reliable fluorescence measurements in microplate format.

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Lab products found in correlation

Calcein am dye, by Thermo Fisher Scientific (2 mentions) Cytosol extraction buffer, by Abcam (1 mentions) Mitotracker green fm, by Thermo Fisher Scientific (1 mentions) 2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)

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Microplate reader (6925 citations) FL600 (52 citations) FL600 microplate fluorescence reader (23 citations) FL600 microplate fluorescent reader (3 citations)

10 protocols using fl600 microplate reader

1

Salivary MMP-8 Quantification by ELISA

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Salivary MMP-8 was measured using ELISA (Quantikine, Biotechne) according to the manufacturer’s instructions. Samples were analysed in duplicate, absorbance read at 450 nm on a FL600 Microplate Reader (Biotek, Swindon, UK) and MMP-8 concentrations calculated from standards by means of a 4-parameter logistic curve fit using the proprietary software (KC4 KinetCalc, Biotek).
Taylor J.J., Jaedicke K.M., van de Merwe R.C., Bissett S.M., Landsdowne N., Whall K.M., Pickering K., Thornton V., Lawson V., Yatsuda H., Kogai T., Shah D., Athey D, & Preshaw P.M. (2019). A Prototype Antibody-based Biosensor for Measurement of Salivary MMP-8 in Periodontitis using Surface Acoustic Wave Technology. Scientific Reports, 9, 11034.
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2

Evaluating Cell Viability in HT-22 Cells

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Cell viability was determined in HT-22 cells by the calcein AM cell viability assay [17 (link)]. HT-22 cells were seeded for 24 h before initiation of the experiment at a density of 5000 cells per well in 96-well plates. Insult cells with 3.0 mM glutamate induce 50–75% cell death and co-treat with IRN and M1–M4 at the concentrations of 100 nM, 1 µM, 10 µM, 50 µM and 100 µM, and 2-(1-adamantyl)-4-methylestrone (ZYC26) at the concentrations of 100 nm, 1 µM, 5 µM and 10 µM for 24 h. After exposure to various treatment paradigms, cells were rinsed with PBS, and cell viability was measured using the membrane-permeant calcein-AM dye (Molecular Probes, Eugene, OR, USA). Calcein-AM is a fluorogenic esterase substrate that is hydrolyzed to a fluorescent product in cells having esterase activity and intact membranes. Cells were incubated in a solution of 1 µM calcein-AM in PBS at 37 °C in the dark. Twenty minutes later, fluorescence was determined using a Bio-Tek (Winooski, VT, USA) FL600 microplate reader with an excitation/emission filter set of 485/530 nm. The results, obtained in relative fluorescent units, are expressed as the percentage of untreated control values.
Chen F., Qi W., Sun J., Simpkins J.W, & Yuan D. (2014). Urinary metabolites of isorhynchophylline in rats and their neuroprotective activities in the HT22 cell assay. Fitoterapia, 97, 156-163.
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3

Evaluating Cell Viability in HT22 Cells

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Cell viability was determined in HT22 cells by the calcein AM cell viability assay [42 (link)]. In brief, HT22 cells were seeded 24 h before initiation of the experiment at a density of 5000 cells per well in 96-well plates and then treated with various test sample solutions with 3 mM glutamate which induced 50–75% cell death for 24 h. After exposure to various treatment paradigms, cells were rinsed with PBS, and cell viability was measured using the membrane-permeant calcein-AM dye (Molecular Probes). Cells were incubated in a solution of 1 μM calcein-AM in PBS at 37°C in dark. Twenty minutes later, fluorescence was determined using a Bio-Tek FL600 microplate reader with an excitation/emission filter set of 485/530 nm. The results, obtained in relative fluorescent units, are expressed as the percentage of untreated control values.
Qi W., Chen F., Sun J., Simpkins J.W, & Yuan D. (2014). Isolation and Identification of Twelve Metabolites of Isocorynoxeine in Rat Urine and their Neuroprotective Activities in HT22 Cell Assay. Planta medica, 81(1), 46-55.
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4

ATP Quantification in Gene-Manipulated Cells

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After treatment (gene transfection with shRNA-TFAM or exposure to EtBr), HEp-2 or HNE2 cells were cultured in six-well plates at a concentration of 5 × 105 cells/well. The cells were trypsinized and collected by centrifugation at 600 × g for 5 min and washed three times with PBS. The transfected cells were sorted by flow cytometry as described in the section on flow cytometry and cell sorting.
The cells were lysed with ATP-releasing buffer containing 100 mM Tris buffer (titrated to pH 7.8 with acetic acid), 2 mM EDTA, and 1% Triton X-100. A total of 5 μl of the lysate was taken for protein determination and another 5 μl of the lysate was added to a 96-well plate. ATP concentrations in the lysates were quantified in triplicate using an ATP determination kit (luciferase-luciferin; Life Technologies) in a BioTek FL600 microplate reader according to the manufacturer's instructions (results were adjusted according to the protein concentration).
Mei H., Sun S., Bai Y., Chen Y., Chai R, & Li H. (2015). Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs. Cell Death & Disease, 6(4), e1710-.
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5

Mitochondria Isolation from Cells

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Cells were trypsinized, collected by centrifugation at 600 × g for 5 min at 4 °C, washed with ice cold PBS, and resuspended in 500 μl of Cytosol Extraction Buffer (BioVision, San Francisco, CA, USA). After incubation on ice for 10 min, the cells were homogenized in an ice cold dounce tissue grinder and transferred to a 1.5-ml tube and centrifuged at 700 × g for 10 min at 4 °C. The supernatant was transferred to a fresh 1.5 ml tube and centrifuged at 10 000 × g for 30 min at 4 °C. The supernatant was removed and the pellet was resuspended in 500 μl Cytosol Extraction Buffer and centrifuged again at 10 000 × g for 30 min at 4 °C. The remaining pellet consisted of mitochondria that were lysed for western blot or labeling with MitoTracker Green FM (Life Technologies) for the analysis of mitochondrial mass in a BioTek FL600 microplate reader.
Mei H., Sun S., Bai Y., Chen Y., Chai R, & Li H. (2015). Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs. Cell Death & Disease, 6(4), e1710-.
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6

Investigating Metabolic Consequences of CcO Inhibition

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This assay was conducted to determine the cellular consequence of CcO inhibition under a hyperexcitatory milieu (glutamate or EW), and to determine whether CcO inhibition blunts the protective effect of E2 on cells. The membrane-permeant Calcein-AM ester dye (Invitrogen, Carlsbad, CA) was used to measure cell viability. Briefly, HT22 cells received the aforementioned EW with or without treatment with NaN3 (1 μM) and/or E2 (1 μM) during EW phases. Separately, HT22 cells were exposed to glutamate (3 mM) for 24 hours with or without NaN3 (1 μM) cotreatment. After the removal of the medium from the 96-well cell plates, the cells were rinsed once with PBS, and incubated in PBS solution containing 2.5 μM Calcein-AM. Twenty minutes later, fluorescence was determined using a BioTek FL600 microplate reader (BioTek Instruments, Winooski, VT) with an excitation/emission filter set at 495/515 nm. Wells treated with methanol served as blanks.
Jung M.E, & Metzger D.B. (2016). A sex difference in oxidative stress and behavioral suppression induced by ethanol withdrawal in rats. Behavioural brain research, 314, 199-214.
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7

Cytotoxicity Profiling of Polyplexes

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The cytotoxicity of lyophilized polyplexes was determined using calcein AM staining to detect live cells. In vitro transfection experiments were carried out as described above. 24 h after transfection, media were aspirated and replaced with 200 μl of a 1 μg/ml calcein AM solution in DPBS. After incubating 30 min at 37 °C, fluorescence intensity was measured using an FL600 microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 530 nm (Bio-Tek Instruments, Winooski, Vermont).
Adolph E.J., Nelson C.E., Werfel T.A., Guo R., Davidson J.M., Guelcher S.A, & Duvall C.L. (2014). Enhanced Performance of Plasmid DNA Polyplexes Stabilized by a Combination of Core Hydrophobicity and Surface PEGylation. Journal of materials chemistry. B, Materials for biology and medicine, 2(46), 8154-8164.
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8

Measuring Intracellular ROS Levels

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Levels of intracellular ROS were assessed using the radical-sensitive dye H2DCFDA, which is oxidized to the fluorescent 2, 7-dicholorofluorescein (DCF) upon exposure to ROS. Following treatment with indicated reagents, the cells were washed with Hank's Balanced Saline Solution (HBSS) twice. Dye was added at a concentration of 10 μM H2DCFDA and incubated for 1 h at 37 °C in darkness. Fluorescence was measured using the excitation/emission wavelengths 485/530 nm with a Biotek FL600 microplate reader.
Egnatchik R.A., Leamy A.K., Jacobson D.A., Shiota M, & Young J.D. (2014). ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload. Molecular Metabolism, 3(5), 544-553.
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9

Quantification of rhBMP-2 Release from Scaffolds

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The protein release from rhBMP2-CHI and rhBMP2-CPS-CHI scaffolds were analyzed at eight different time points (0.5, 1, 2, 3, 4, 5, 120, 216 hours) using specific sandwich ELISA assay (Peprotech, UK) and following manufacturer instructions. Briefly, a 96-well microplate was precoated with a rabbit anti-BMP-2 antibody overnight and blocked over 1 hour with 1% BSA in PBS 1X pH 7.4. Afterwards, 100 µL of culture medium was placed into the microplate and incubated over 2 hours. Then, biotinylated rabbit anti-BMP-2 antibody was added and incubated over an additional hour. After that, an Avidin-HRP conjugated was added to the microplate and incubated over 30 minutes. Finally, ABTS (Sigma) was added and the absorbance at 405 nm was measured using a Biotek FL-600 microplate reader. All the process was carried out at room temperature using the absorbance of a culture medium non-exposed to scaffolds as blank readout. Data obtained were converted to ng/mL by interpolation on standard curve. Total rhBMP-2 content was determined multiplying by culture medium total volume (400 µL).
Guzmán R., Nardecchia S., Gutiérrez M.C., Ferrer M.L., Ramos V., del Monte F., Abarrategi A, & López-Lacomba J.L. (2014). Chitosan Scaffolds Containing Calcium Phosphate Salts and rhBMP-2: In Vitro and In Vivo Testing for Bone Tissue Regeneration. PLoS ONE, 9(2), e87149.
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10

Biofilm Quantification of P. aeruginosa

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P. aeruginosa PAO1 enrichments were transferred into 96-well microtiter plates at an optical density (OD) of 0.03 at 600 nm. Wells were supplemented with TSB (1× final concentration) and the agents shown in Table 1. Eight replicates per agent and control were tested. Plates were incubated at 37 °C for 48 h. Quantitative analysis in microtiter plates was performed following the crystal violet method [35] . At selected time points (i.e., 0, 24, 48 h), plates were washed five times in deionized water (DIW) to remove planktonic cells. Wells were stained with 300 µL of 1:3 diluted crystal violet solution for 20 min at 22 °C. Excess stain was washed off five times in DIW. Following washing, 300 µL of 95% ethanol was added to each well, and de-colorization of the wells was allowed for 15 min. Light absorbance data were read at 540 nm in a BioTek FL600 micro-plate reader.
Özcan S.S., Dieser M., Parker A.E., Balasubramanian N, & Foreman C.M. (2019). Quorum sensing inhibition as a promising method to control biofilm growth in metalworking fluids. Journal of industrial microbiology & biotechnology, 46(8).
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