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M200 pro spectrophotometer

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The M200 Pro spectrophotometer is a compact, high-performance instrument designed for a wide range of applications in life science laboratories. It provides precise and reliable absorbance measurements across a broad wavelength range.

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Lab products found in correlation

Anti m13 hrp conjugate, by Sino Biological (3 mentions) Tmb substrate, by Avantor (3 mentions) Nunc maxisorp flat bottom clear plates, by Thermo Fisher Scientific (3 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Amphotericin b, by Thermo Fisher Scientific (1 mentions) Qiashredder, by Qiagen (1 mentions) Rlt buffer, by Qiagen (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Nanoquant plate, by Tecan (1 mentions) Mv4 11 cells, by American Type Culture Collection (1 mentions)

15 protocols using m200 pro spectrophotometer

1

Outer Membrane Permeability Assay

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Outer membrane permeabilization was measured using nitrocefin as a chromogenic substrate of periplasmic -lactamase. Ten milliliters of MH broth were inoculated with 0.1 mL of an overnight culture of EA289 bacteria and grown at 37°C until the OD600 nm reached 0.5. The remaining steps were performed at room temperature. Cells were recovered by centrifugation (4000 rpm for 20 min) and washed once in 20 mM potassium phosphate buffer (pH 7.2) containing MgCl2 (1 mM). After a second centrifugation, the pellet was re-suspended and adjusted to a OD600 nm to 0.5. Then, 50 µL of either Polymyxin B (positive control) or the A1 compound were added to 100 µL of the cell suspension to obtain a final concentration varying from 0.98 µM to 500 µM. Fifty microliters of nitrocefin were then added to obtain a final concentration of 50 µg/mL. Nitrocefin hydrolysis was monitored spectrophotometrically by measuring the increase in absorbance at 490 nm. Assays were performed in 96-well plates using a M200 Pro Tecan spectrophotometer.
Blanchet M., Borselli D., Rodallec A., Peiretti F., Vidal N., Bolla J.M., Digiorgio C., Morrison K.R., Wuest W.M, & Brunel J.M. (2018). Claramines: A New Class Of Broad-Spectrum Antimicrobial Agents With Bimodal Activity. ChemMedChem, 13(10).
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2

Outer Membrane Permeability Assay

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Outer membrane permeabilization was measured using nitrocefin as a chromogenic substrate of periplasmic -lactamase. Ten milliliters of MH broth were inoculated with 0.1 mL of an overnight culture of EA289 bacteria and grown at 37°C until the OD600 nm reached 0.5. The remaining steps were performed at room temperature. Cells were recovered by centrifugation (4000 rpm for 20 min) and washed once in 20 mM potassium phosphate buffer (pH 7.2) containing MgCl2 (1 mM). After a second centrifugation, the pellet was re-suspended and adjusted to a OD600 nm to 0.5. Then, 50 µL of either Polymyxin B (positive control) or the A1 compound were added to 100 µL of the cell suspension to obtain a final concentration varying from 0.98 µM to 500 µM. Fifty microliters of nitrocefin were then added to obtain a final concentration of 50 µg/mL. Nitrocefin hydrolysis was monitored spectrophotometrically by measuring the increase in absorbance at 490 nm. Assays were performed in 96-well plates using a M200 Pro Tecan spectrophotometer.
Spitz C., Mathias F., Péchiné S., Doan T.H.D., Innocent J., Pellissier S., Di Giorgio C., Crozet M.D., Janoir C, & Vanelle P. (2019). 2,4-Disubstituted 5-Nitroimidazoles Potent against Clostridium difficile. ChemMedChem, 14(5).
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3

Cell Viability Assay for MV4-11 Cells

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MV4–11 cells were maintained in IMDM medium containing 10% FBS at 37 °C in a 5% CO2 humidified incubator. Cells were plated (50,000 per well) in flat-bottom 24-well plates and allowed to grow for 12 h, then CellTiter-Blue was added to selected wells to a final concentration of 0.125 mg/mL to measure the cell viability at the time of inhibitor addition (t = 0), and was incubated for 2–4 h until sufficient color changed occurred. At the same time, the remaining wells were treated with 13a (200 nM), 13a (200 nM) with z-VAD (20 μM), chloroquine (5 μM) or wortmannin (100 nM) for 24 h. CellTiter-Blue was added to each well to a final concentration of 0.125 mg/mL. The mixture was allowed to incubate for the same time as the t = 0 wells. Cell viability was measured as a function of resorufin fluorescence intensity using a Tecan M200 Pro spectrophotometer, 560 nm/590 nm (excitation/emission). Data were normalized to control wells and background was removed. Cell viability with each treatment was compared with t = 0.
Li X., Jiang Y., Peterson Y.K., Xu T., Himes R.A., Luo X., Yin G., Inks E.S., Dolloff N., Halene S., Chan S.S, & Chou C.J. (2020). Design of hydrazide-bearing HDACIs based on panobinostat and their p53 and FLT3-ITD dependency in anti-leukemia activity. Journal of medicinal chemistry, 63(10), 5501-5525.
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4

Cell Viability Assay of HT-22 Neurons

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HT-22 neuronal cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM/high glucose) with 10% fetal bovine serum (FBS) and 1% of antibiotic-antimycotic (Amphotericin B, Penicillin, and Streptomycin; Invitrogen) at 37 °C in 5% CO2. 2000 cells per well were plated in a clear 96-well plate, 12 h later, serially diluted compounds were added and incubated for 48 h. CellTiter-Blue (resazurin cell viability assay reagent) was added at a final concentration of 0.125 mg/mL to each well. The cell viability was determined after 2 h via resorufin fluorescence intensity at ex 560 nm/em 590 nm using a Tecan M200 Pro spectrophotometer. Data were processed and fitted via Prism (GraphPad) by subtracting the background and normalizing to the control wells.
Li X., Himes R.A., Prosser L.C., Christie C.F., Watt E., Edwards S.F., Metcalf C.S., West P.J., Wilcox K.S., Chan S.S, & Chou C.J. (2020). Discovery of the first Vitamin K analog as a potential treatment of pharmacoresistant seizures. Journal of medicinal chemistry, 63(11), 5865-5878.
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5

Microarray Analysis of Rat RNA

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Frozen samples were pulverized with the help of mortar and pestle cooled with liquid nitrogen. The samples were transferred to RLT buffer (Qiagen, Hilden, Germany) with 1% β-mercaptoethanol. The RNA was extracted according to the manufacturer’s instructions via the RNeasy mini kit (Qiagen, Hilden, Germany) with the additional use of QiaShredder (Qiagen, Hilden, Germany). Quality and quantity of the RNA were checked with an Agilent 2100 Bioanalyzer with an RNA Nano Lab chip and a Tecan M200 Pro spectrophotometer with a NanoQuant Plate at 260 nm and 280 nm. For the quality measurements, it was verified that the samples had RNA integrity numbers (RIN) higher than nine as well as A260/A280 ratios of 1.8–2 and A260/A230 ratios higher than two. 300 ng RNA of each sample was shipped. The samples were labeled with a GeneChip WT Plus labeling kit at Eurofins (Aarhus, Denmark, Comp. No. 7230-GT0028). The microarray analysis on Affymetrix Clariom S rat array (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was performed at Eurofins Genomics Europe Genotyping A/S (Eurofins, Aarhus, Denmark). The raw data of this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE166195.
Buchmueller J., Sprenger H., Ebmeyer J., Rasinger J.D., Creutzenberg O., Schaudien D., Hengstler J.G., Guenther G., Braeuning A, & Hessel-Pras S. (2021). Pyrrolizidine alkaloid-induced transcriptomic changes in rat lungs in a 28-day subacute feeding study. Archives of Toxicology, 95(8), 2785-2796.
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6

Cytotoxicity Assay for Leukemia Cells

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MV4–11 (20,000 per well) or primary leukemia (100,000 per well) cells were plated in 96-well clear plates and pre-incubated overnight, then serially diluted inhibitors (3 times dilution) with 12 concentrations from 5 μM were added. Cells were incubated with compounds for 48 h. CellTiter-Blue was added to a final concentration of 0.125 mg/mL. The mixture was allowed to incubate for 2–4 h until sufficient color changed occurred. Cell viability was measured as a function of resorufin fluorescence intensity using a Tecan M200 Pro spectrophotometer, 560 nm/590 nm (excitation/emission). Data were normalized to control wells and background was removed. EC50s were determined using GraphPad Prism’s “log(inhibitor) vs. normalized response-variable slope” function.
Li X., Jiang Y., Peterson Y.K., Xu T., Himes R.A., Luo X., Yin G., Inks E.S., Dolloff N., Halene S., Chan S.S, & Chou C.J. (2020). Design of hydrazide-bearing HDACIs based on panobinostat and their p53 and FLT3-ITD dependency in anti-leukemia activity. Journal of medicinal chemistry, 63(10), 5501-5525.
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7

Determination of SARS-CoV-2 Spike Neutralization

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HEK-ACE2 were seeded at 10,000 cells/well in 40 μL of D10 on 96-well white plates (Corning, cat. 354620) 24 hours prior to infection. To determine IC50 for pseudotyped virus, dose series of each VH binder were prepared at 3x concentration in D10 media and 50 μL were aliquoted into each well in 96-well plate format. Next, 50 μL of virus diluted in D10 media were added to each well and the virus and blocker solution was allowed to incubate for 1 hr at 37°C. Subsequently, 80 μL of the virus and blocker solution were transferred to wells seeded with HEK-ACE2. After 60 hrs of infection at 37°C, intracellular luciferase signal was measured using the Bright-Glo™ Luciferase Assay. 80 μL of reconstituted Bright-Glo™ luciferase reagent was added to each well, incubated at room temperature with gentle shaking for 5 min, before the luminescence was measured on a Tecan M200 pro spectrophotometer. The half-maximal inhibitory concentration (IC50) was determined using a 4-parameter nonlinear regression (GraphPad Prism).
Bracken C.J., Lim S.A., Solomon P., Rettko N.J., Nguyen D.P., Zha B.S., Schaefer K., Byrnes J.R., Zhou J., Lui I., Liu J., Pance K., Zhou X.X., Leung K.K, & Wells J.A. (2020). Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2. Nature chemical biology, 17(1), 113-121.
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8

Combination Cytotoxicity Assay of Inhibitors

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20,000 MV4–11 cells were plated per well in 96-well clear plates and pre-incubated overnight, then serially diluted 13a and RG7388, bortezomib, or 17-AAG were added alone and combined. For the experiment for 13a and RG7388, concentration of 13a was 25 nM, RG7388 was 2 times dilution from 500 nM. For the experiment of 13a and bortezomib, concentrations of 13a were 405 nM, 135 nM, 45 nM, 15 nM, and 5 nM, respectively, while concentration of bortezomib was 8 nM. In the experiment of 13a in combination with 17-AAG, final comcentration of 13a was 9.87 nM, concentrations of 17-AAG were 300 nM, 200 nM, 133 nM, 88 nM, and 59 nM, respectively. Then cells were incubated for 24 h (RG7388 and bortezomib) and 48 h (17-AAG), CellTiter-Blue was added to each well to a final concentration of 0.125 mg/mL. The mixture was allowed to incubate for 2–4 h until sufficient color changed occurred. Cell viability was measured as a function of resorufin fluorescence intensity using a Tecan M200 Pro spectrophotometer, 560 nm/590 nm (excitation/emission). The combination index (CI) was calculated using CompuSyn software.
Li X., Jiang Y., Peterson Y.K., Xu T., Himes R.A., Luo X., Yin G., Inks E.S., Dolloff N., Halene S., Chan S.S, & Chou C.J. (2020). Design of hydrazide-bearing HDACIs based on panobinostat and their p53 and FLT3-ITD dependency in anti-leukemia activity. Journal of medicinal chemistry, 63(10), 5501-5525.
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9

Cytotoxicity Assay for PBMCs

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Human PBMCs were graciously donated from Dr. Nathan Dolloff s laboratory. Cells were flash thawed from liquid nitrogen using RPMI-1640 media + Glutamax and 15% Fetal Bovine Serum and allowed to incubate overnight at 37°C, 5% CO2. Cells were centrifuged at 1000 RPM for 5 minutes. Pelleted, healthy cells were reseeded at 50k cells/well and treated immediately with serially diluted inhibitors (diluted in medium). Cells were allowed to incubate with inhibitor or vehicle for 24 hours before addition of 0.125 mg/mL (final) CellTiter-Blue. The mixture was allowed to incubate until sufficient color changed occurred. Cell viability was measured as a function of resorufin intensity using a Tecan M200 Pro spectrophotometer, 560 nm (ex.)/590 nm (em.). Data were normalized to control wells and background was subtracted.
McClure J.J., Zhang C., Inks E.S., Peterson Y., Li J, & Chou C.J. (2016). Development of Allosteric Hydrazide-Containing Class I Histone Deacetylase Inhibitors for Use in Acute Myeloid Leukemia. Journal of medicinal chemistry, 59(21), 9942-9959.
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10

Phage Display Screening for Spike-RBD Binders

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For each phage clone, 4 different conditions were tested—Direct: Spike-RBD-Fc, Competition: Spike-RBD-Fc with an equal concentration of Spike-RBD-Fc in solution, Negative selection: ACE2-Fc/Spike-RBD-Fc complex, and Control: Fc. 384-well Nunc Maxisorp flat-bottom clear plates (Thermo Fisher Scientific) were coated with 0.5 μg/mL of NeutrAvidin in PBS overnight at 4°C and subsequently blocked with PBS + 0.02% Tween-20 + 2% BSA for 1 hr at room temperature. Plates were washed 3X with PBS containing 0.05% Tween-20 (PBST) and were washed similarly between each of the steps. 20 nM of biotinylated Spike-RBD-Fc, ACE2-Fc/Spike-RBD-Fc complex, or Fc diluted in PBSTB was captured on the NeutrAvidin-coated wells for 30 min, then blocked with PBSTB + 10 μM biotin for 30 min. Phage supernatant diluted 1:5 in PBSTB were added for 20 min. For the competition samples, the phage supernatant was diluted into PBSTB with 20 nM Spike-RBD-Fc. Bound phage were detected by incubation with anti-M13-HRP conjugate (Sino Biological)(1:5000) for 30 min, followed by the addition of TMB substrate (VWR International). The reaction was quenched with the addition of 1 M phosphoric acid and the absorbance at 450 nm was measured using a Tecan M200 Pro spectrophotometer.
Bracken C.J., Lim S.A., Solomon P., Rettko N.J., Nguyen D.P., Zha B.S., Schaefer K., Byrnes J.R., Zhou J., Lui I., Liu J., Pance K., Zhou X.X., Leung K.K, & Wells J.A. (2020). Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2. Nature chemical biology, 17(1), 113-121.
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