Spectra max m5

Manufactured by Agilent Technologies

Sourced in United States

The SpectraMax M5 is a multimode microplate reader designed for a variety of applications in life science research. It measures absorbance, fluorescence, and luminescence in microplate samples. The instrument features a xenon flash lamp, dual-grating monochromator, and sensitive photomultiplier tube detector to provide accurate and reproducible results.

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Lab products found in correlation

Sytox green, by Thermo Fisher Scientific (3 mentions) Synergy 4, by Agilent Technologies (2 mentions) Multicycle software, by Phoenix Pharmaceuticals (1 mentions) Cck 8 assay, by Beyotime (1 mentions) Transferrin, by Merck Group (1 mentions) Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions) Crystal violet, by Beyotime (1 mentions) Paraformaldehyde (pfa), by Beyotime (1 mentions) Cytotox 96 kit, by Promega (1 mentions) Evos fl auto, by Thermo Fisher Scientific (1 mentions) Intracellular ph assay kit, by Merck Group (1 mentions) Cytation 5, by Agilent Technologies (1 mentions) Synergy ht, by Agilent Technologies (1 mentions) D300e digital dispenser, by Tecan (1 mentions)

15 protocols using spectra max m5

Cell Cycle and Apoptosis Quantification

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The effects of RuPOP and/or TRAIL on the cell cycle progression and the induction of apoptotic cell death were quantified by flow cytometric analysis. Briefly, treated or untreated cells were trypsinized, washed with PBS and fixed with 70% ethanol overnight at −20°C. The fixed cells were washed with PBS and incubated with a PI working solution for 4 h in darkness. The stained cells were analyzed with flow cytometer (Beckman Coulter, Fullerton, CA). Cell cycle distribution was analyzed using MultiCycle software (Phoenix Flow Systems, San Diego, CA). The proportion of cells in G0/G1, S, and G2/M phases was represented as DNA histograms. Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. For each experiment, over 10000 events per sample were recorded.
Caspase activity was detected by fluorescence assay using specific substrates. Briefly, cell lysates were placed in 96-well plates and then the specific caspase substrates were added. Plates were incubated at 37°C for 2 h and caspase activity was determined by measuring the fluorescence intensity on a microplate spectrophotometer (Spectra Max M5, Bio-Tek) with the excitation and emission wavelengths set at 380 and 440 nm, respectively.

Cao W., Zheng W, & Chen T. (2015). Ruthenium Polypyridyl Complex Inhibits Growth and Metastasis of Breast Cancer Cells by Suppressing FAK signaling with Enhancement of TRAIL-induced Apoptosis. Scientific Reports, 5, 9157.

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CCK8 Assay for Cell Proliferation

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The CCK8 assay (Beyotime, China) was performed to assess cell proliferation. The cells were adjusted to 2 × 10⁵ cells/well, seeded into a 96-well plate at 24 h after transfection, and cultured for different times (24, 48, and 72 h). Then, 10 μl CCK8 solution was added to each well, followed by incubation for 1.5 h at 37°C. The optical density value at 450 nm was measured using a SpectraMax M5 ELISA plate reader (BioTek, Winooski, VT, U.S.A.).

Zhou W., Wang H., Yang J., Long W., Zhang B., Liu J, & Yu B. (2019). Down-regulated circPAPPA suppresses the proliferation and invasion of trophoblast cells via the miR-384/STAT3 pathway. Bioscience Reports, 39(9), BSR20191965.

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Cellular Uptake of RuPOP Measured

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Cells were cultured in 96-well plates for 24 h. Then 0.25–2 mg of transferrin (Sigma-Aldrich) were added to the wells for 1 h, followed by the co-treatment of RuPOP (20 μM) for 6 h. After that, the cells were washed with PBS and lysed by 100 ml/well of Triton X-100(1%) in 0.1 M NaOH solution, followed by the fluorescence intensity measurement of the internalized RuPOP (Spectra Max M5, Bio-Tek).

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Intracellular Uptake of RuPOP

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The intracellular uptake of RuPOP was determined by using a microplate spectrophotometer (Spectra Max M5, Bio-Tek) according to our previous methods45 (link). Briefly, the treated cells were washed with PBS three times and lysed by 100 μl/well of Triton X-100(1%) in 0.1 M NaOH solution. Then the fluorescence intensity of the internalized RuPOP was detected with excitation and emission wavelengths set at 418 and 590 nm, respectively. The cell number was determined by Trypan blue staining assay, and thus the intracellular RuPOP content was calculated as μM RuPOP per 10⁸ cells.

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Evaluating Cell Proliferation: CCK-8 and Colony Assay

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Cell proliferation was evaluated by Cell Counting Kit‐8 (CCK‐8) assay (Dojindo Laboratories) and colony formation assay. For CCK‐8 assay, 5000 cells per well were first seeded into 96‐well plates. Then, on day 0, 1, 2, 3, and 4, 10 ml of CCK‐8 solution was added to each well. After incubating at 37°C for 3 h, cell growth was measured at 450 nm with Spectra‐Max M5 multi‐functional microplate reader (Synergy 4, Bio‐Tek). For the colony formation assay, 1000 cells per well were first seeded in 6‐well plates, then maintained in complete medium, and after incubation at 37°C for 14 days, cells were fixed with 4% paraformaldehyde (Beyotime) for 30 min and stained with 0.1% crystal violet (Beyotime). After gently washing with PBS and air‐drying, cell colonies were counted and analyzed. The experiment was repeated three times.

Sun W., Zhang H., Duan W., Zhu H, & Gu C. (2021). Tumor suppressor role of hsa_circ_0035445 in gastric cancer. Journal of Clinical Laboratory Analysis, 35(6), e23727.

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Intracellular ROS Detection Assay

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Intracellular free radicals were measured using the Cellular Reactive Oxygen Species Detection Assay commercial kit (Abcam-186027, Cambridge, UK). In this kit, a fluorescence probe that is permeable to cells was used. When the probe reacts with ROS, it produces red fluorescence. According to the kit protocol, the cells were planted in black plates and incubated for 24 h. The compound E in PBS was administered at doses of 0–200 μM and incubated at 37 °C within an atmosphere of 5 % CO₂ for 1 h (protected from light). After the compound E was poured out, the wells were filled with 100 µL of ROS Deep Red Working Solution, and then they were incubated at 37°C under an atmosphere of 5 % CO₂ for 1 h. A fluorometry device (Spectramax M5, BioTek, VT, USA) Ex/Em: 650/675 nm (cut off: 665 nm) was used for the measuring.

Temiz E., Koyuncu I., Durgun M., Caglayan M., Gonel A., Güler E.M., Kocyigit A, & Supuran C.T. (2021). Inhibition of Carbonic Anhydrase IX Promotes Apoptosis through Intracellular pH Level Alterations in Cervical Cancer Cells. International Journal of Molecular Sciences, 22(11), 6098.

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Neutrophil DNA Extrusion Monitoring

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Neutrophils of healthy donors were stimulated using PMA (25 nM) for 2.5 h, and the impermeable DNA dye SYTOX Green (Thermo Fisher, USA, Cat No. S7020) was added to each well at a volume ratio of 1 : 100. Subsequently, fluorescence intensity was measured using a SpectraMax M5 multifunctional microplate reader (Biotek, USA) 10 min later. After PMA stimulation, the fluorescence intensity was recorded every 30 min for a total of 3.5 h.

Han X., Zhang X., Song R., Li S., Zou S., Tan Q., Liu T., Luo S., Wu Z., Jie H, & Wang J. (2023). Necrostatin-1 Alleviates Diffuse Pulmonary Haemorrhage by Preventing the Release of NETs via Inhibiting NE/GSDMD Activation in Murine Lupus. Journal of Immunology Research, 2023, 4743975.

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Cytotoxicity Evaluation of Compound E

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The cytotoxic effects of compound E were evaluated with WST kit (Roche, Basel, Switzerland) in accordance with manufacturer protocols. The cells were planted in 96 well plates (10⁴ cells per well). After 24 h of incubation, compound E at doses of 0, 2.5, 5, 10, 25, 50, 100, and 200 μM was administrated on the cell lines for 24, 48, and 72 h, and 5-FU on the control cell line with the same doses. WST-1 reactive of 10 µL was added in all wells. Following 4 h incubation, the measurements were carried out in a plate reader (Spectramax M5, BioTek, VT, USA) at wavelengths of 450 and 630 nm. Consequently, graphs were created, and the IC₅₀ values of compound E and 5-fluorouracil were calculated. Selectivity index (SI) can be defined as the ratio of the toxic concentration of a sample against its effective compound concentration.

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LDH Release in Cell Assays

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Lactate dehydrogenase (LDH) release from cells was measured in culture supernantants from either the XTT or caspase activation assays. Cells were incubated with the cell lysis solution (Promega Cytotox 96 kit) for 45 minutes to obtain a positive control while untreated cells were used as a negative control. 50μl of supernatant from each well was transferred to a fresh 96-well plate and combined with 50μl of reconstituted Substrate Mix (kit). Plates were incubated for 30 minutes at room temperature for 30 minutes prior to adding 50μl of stop solution (kit). Absorbance was read at 490nm on a Biotek Synergy 4 or SpectraMax M5 plate reader. Absorbance was compared to a reference wavelength (690nm) and background subtracted using wells with test material but no cells.

Moore L., Grobárová V., Shen H., Man H.B., Míčová J., Ledvina M., Štursa J., Nesladek M., Fišerová A, & Ho D. (2014). Comprehensive Interrogation of the Cellular Response to Fluorescent, Detonation and Functionalized Nanodiamonds. Nanoscale, 6(20), 11712-11721.

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Cellular Uptake and Localization of F/A-PLGA@DOX/SPIO

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A549 and L02 cells were inoculated at the density of 10 × 10⁴ cells/mL into a 96-pore plate to be cultivated overnight. 0.5 mg/mL of DOX and F/A-PLGA@DOX/SPIO were added to the incubator for different durations; culture media were collected at 0, 0.5, 1, 2, 4, and 6 h, and a mixture of 100 μL HCl·DMSO (1:4) was added to dissolve cells. Then, F/A-PLGA@DOX/SPIO and DOX solutions in the same block were diluted to equal ratios according to their concentrations. The light absorption of each pore was detected via fluorescence microplate (Spectra Max M5, BioTek, Winooski, VT). The amount of DOX and F/A-PLGA@DOX/SPIO absorbed by the cells was calculated at different times through the standard curve.
A Lyso-tracker was employed to track the position of F/A-PLGA@DOX/SPIO in the A549 cells. First, A549 cells were cultivated at a density of 5 × 10⁴ cells/mL in the 2-cm-thick culture medium. After 24 h, F/A-PLGA@DOX/SPIO of a certain concentration was added, incubated for 8 h, and disposed at 0, 1, 2, 4, 6, and 8 h. Later, the green lysosomal marker, Lyso-tracker (1 μg/ml), and nucleus blue marker, Hoechst 33342 (0.1 μg/mL), were used to incubate cells for 1.5 h and 30 min, respectively. Finally, we collected fluorescence cell images using a fluorescence microscope (EVOS FL Auto, Life Technologies, 20×,Walsham, MA, USA).

Gao P., Mei C., He L., Xiao Z., Chan L., Zhang D., Shi C., Chen T, & Luo L. (2018). Designing multifunctional cancer-targeted nanosystem for magnetic resonance molecular imaging-guided theranostics of lung cancer. Drug Delivery, 25(1), 1811-1825.

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