Streptavidin-coated 96-well plates (Thermo Scientific) were used to study the impact of organic acids on liposomes internal pH. Wells were washed with 10 mM Na-HEPES buffer (pH 7.4). After, 50 μl of biotinylated liposomes were added to the wells and incubated at room temperature for 30 min, with shaking. After the binding step, wells were washed with 10 mM Na-HEPES buffer (pH 7.4), to remove unbound liposomes and traces of pyranine. The fluorescence (emission at 510 nm) of the bound liposomes was measured in the M200 plate reader (Tecan) and the ratio between excitation 453 nm and 405 nm was converted to pH using the above equation. Next, organic acid solutions, 65 mM at different pH values, were added to the wells containing the liposomes. The pH of the organic acids solutions was adjusted to the desired pH by the addition of KOH or HCl 1 M. Incubation with organic acids was done at room temperature for 5 min, with shaking. We tested different incubation times (10, 20, and 30 min, data not shown). It turned out that 5 min are enough for the organic acid diffusion to occur. Therefore, this was the time chosen to perform the study. Finally, M200 plate reader (Tecan) was used to monitor the fluorescence intensity changes caused by the addition of organic acids to the wells. Final pH values inside the liposomes were obtained using the equation.
M200 plate reader
Manufactured by Tecan
Sourced in Switzerland
The M200 plate reader is a compact and versatile instrument designed for a wide range of absorbance-based assays. It features a wavelength range of 200 to 1000 nm and can accommodate 6- to 384-well microplates. The M200 provides accurate and reproducible results, making it suitable for a variety of applications in life science research and clinical laboratories.
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Lab products found in correlation
Cyquant direct cell proliferation assay, by Thermo Fisher Scientific (6 mentions)
Costar 96 well elisa plates, by Corning (2 mentions)
Prism 6, by GraphPad (2 mentions)
Streptavidin coated 96 well plate, by Thermo Fisher Scientific (1 mentions)
Elisa max deluxe, by BioLegend (1 mentions)
Jurkat, by American Type Culture Collection (1 mentions)
Hl 60, by American Type Culture Collection (1 mentions)
Mycoalert, by Lonza (1 mentions)
Upa activity assay kit, by Merck Group (1 mentions)
Akt inhibitor mk 2206, by Santa Cruz Biotechnology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Percoll plus, by GE Healthcare (1 mentions)
Cholesterol oxidase, by Merck Group (1 mentions)
F1303, by Thermo Fisher Scientific (1 mentions)
Rotitest vital, by Carl Roth (1 mentions)
96 well plate, by Corning (1 mentions)
Breathe easy sealing membrane, by Merck Group (1 mentions)
Sigmaplot 10, by Merck Group (1 mentions)
69 protocols using m200 plate reader
1
Organic Acids Impact on Liposome pH
2
Quantification of Cytokine Levels in Human Plasma
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Detection of total / overall amounts of IFNγ and IL2 in human plasma was conducted using ELISA MAX Deluxe sets from Biolegend, Germany: IFNγ (#430106) and IL2 (#431806). The manufacturer’s Avidin-horseradish peroxidase conjugate was replaced by PolyHRP80 streptavidine conjugate (#SP80C, SDT Reagents, Germany) in order to achieve a tenfold lower limit of detection. Lower limit of detection (Background + 3x S.D.) was generally at 2 – 5 pg/ml for IFNγ and IL2, respectively. Initial dilution of control and test samples was performed described as follows: negative control (NaCl) 1/5, 1st positive control (SEB) 1/2500 for IFNγ and 1/500 for IL2, 2nd positive control (CEFT) 1/50 for IFNγ and 1/25 for IL2, test samples (HBcAg and HBsAg) 1/5. If a test sample’s absorbance value fell outside the maximum standard curve range, these samples were subsequently retested with a tenfold higher dilution, e.g. 1/5 → 1/50, 1/500 → 1/5000, 1/2500 → 1/25000. A seven-point standard curve from 1 – 64 pg/ml IFNγ or IL2 was used for quantitation. Standards, controls and test samples were measured in duplicate. The samples were analyzed using Magellan software (version 6.5) equipped on a Tecan M200 plate reader.
3
Modulating Cancer Cell Signaling Pathways
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Cancer cell lines PC3, SIHA, Jurkat and HL-60 cells were obtained from the American Type Culture Collection (ATCC) and regularly tested to ensure the absence of Mycoplasma contamination (MycoAlert, Lonza). New stock vials were thawed every 3–4 months and cell morphology regularly checked. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37°C with 5% CO2. PN1 expression vectors were transfected into cells using FuGENE 6 reagent (Roche). All transfection experiments performed for 24 h unless otherwise noted. Where indicated, inhibitors, recombinant proteins, or blocking antibodies were added to serum free medium during treatments: NF-κB inhibitor Bay11–7082 (BIOMOL Research laboratories, PA), PI3k/AKT inhibitor LY 294002 (Calbiochem), AKT inhibitor MK-2206 (Santa Cruz), recombinant uPA (America Diagnostica), recombinant PN1 (R&D system), uPAR blocking antibody (R&D Systems), and LRP blocking antibody (Progen, Germany). Recombinant human TRAIL was generated as described [45 (link)]. uPA activity was measured using a uPA Activity Assay kit (Millipore), per manufacturer's instructions. Measurements were taken at 405 nM on a Tecan m200 plate reader.
4
Evaluating RANK Expression in Pre-Osteoclasts
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To measure the possible direct effect of RvD1 on pre-osteoclast cells, we chose to evaluate the RANK expression on RAW cells w/o RvD1treatment by using a PE- anti mouse CD265 (RANK) IF Abs. In brief, following 1 and 3 days of RANKL incubation, cells were fixed with 4% PFA for 10 minutes, stained with 1% CD265 antibody for 45 minutes, and counterstained with 1% DAPI for 5 minutes. RANK expression was measured with a florescent plate reader (Tecan© M200 Plate Reader). To visualize, digital images were also taken using a fluorescence microscope (Nikon TL, Tochigi, Japan).
5
Apoptosis Quantification and Caspase Activation
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Apoptosis was measured using PE annexin V Apoptosis Detection Kit I (BD Biosciences, Austria). Caspase-8 and caspase-9 activation was determined using the Milliplex human early apoptosis kit (Millipore, Germany). For MTT assays, cells were incubated with 0.5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich, Austria) for 1 h at 37 °C in the dark. Cells were lysed using isopropylalcohol containing 0.1% NP-40 and 0.04 N HCl. Absorbance was read at 565 nm in a Tecan M200 plate reader.
6
Screening GFP Expressing Pichia Clones
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Pichia colonies expressing GFP variants were picked in triplicate into standard V-bottom 96-well microtiter plates containing 0.15 mL YNB medium and 20 g L−1 glucose. Cultures were grown overnight and diluted into 0.8 mL fresh YNB medium in deep 96-well plates to an initial OD600 of approximately 0.1. Cultures were incubated with shaking for 12 h prior to OD600 and fluorescence measurements. Fluorescence was measured using the M200 plate reader (Tecan) using an excitation wavelength of 485 nm and an emission wavelength of 525 nm. Gain was adjusted manually for each sample. A strain lacking GFP was included as a control for autofluorescence.
7
Yeast Growth and Stress Response
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Yeast strains were grown in standard rich media YPD or synthetic complete (SC) medium unless otherwise indicated. Serial dilution assays were performed as described previously (Stirling et al., 2011 (link)). In brief, an identical OD of cells was serially diluted tenfold and spotted on the indicated plate with a 48-pin replica pinning manifold and incubated at indicated temperatures for 72 h. Yeast growth curves in YPD media at 30°C were performed as previously described by Stirling et al. (2012) (link), and logarithmic phase cultures treated with or without MMS for 2 h were washed twice and then grown for another 2 h after washout. These were diluted to an OD of 0.05 in a 96-well plate in triplicate and grown for 48 h in an M200 plate reader (TECAN) at 30°C. Fig. 6 B shows the growth curves from six biological replicates for each strain. Standard MMS treatments (unless indicated) were at a concentration of 0.05% for 2 h (∼99%; Sigma-Aldrich). All other chemical treatments were MG132 (80 µl), CHX (200 µg/ml), and rapamycin (200 nM) for 2 h. Table S5 contains a list of yeast strains, including database IDs, genotypes, primers, and plasmids used.
8
Measuring UV-induced Growth Defects
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Wild-type (BY4742) and rad52 Δ yeast strains were diluted from midlog phase to OD600 = 0.01 in 200 µl SC media ± CX-5461 in 96-well plates. Cells were incubated for 3 hr ± CX-5461 with constant shaking before UVA treatment and subsequent loading to a TECAN M200 plate reader. OD600 readings were measured every 30 min over a period of 24 hr and plates were shaken for 10 min before each reading. Strains were tested in three replicates per plate per condition and the area under the curve (AUC) was calculated for each replicate. Strain fitness was defined as the AUC of each yeast strain relative to the AUC of the wild-type strain (without CX-5461 and UVA treatment) grown on the same plate.
9
Investigating Cisplatin Resistance in Yeast
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Yeast strains were derived from the MATa, BY4741 Yeast Knockout Collection [17 (link)]. Overnight cultures were diluted to an optical density at 600 nanometers (OD600) of approximately 0.07 in media with or without 40μM cisplatin (Sigma) and grown in a Tecan M200 plate reader for 24 hours at 30°C. Shaking and OD600 measurements were done every 30 minutes during the growth period. Growth curves were analyzed essentially as described [18 (link),19 (link)]. Briefly, 4 to 6 replicate curves for each condition were generated and the area under each curve was expressed as a proportion of the corresponding wildtype (WT) untreated sample. Multiplying the fitness effect of cisplatin on WT cells by the fitness effect of the indicated mutation created the Expected fitness based on the product or multiplicative model [20 (link)]. The observed and expected values were compared using a T-test.
10
Lipid Quantification in Malaria Parasites
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Each sample was washed three times with TBS [20 mM Tris-HCl, 150 mM NaCl, pH 7.4]. The trophozoite and schizont stages in pRBCs (packed cells) were separated by Percoll PLUS (GE Healthcare, Uppsala, Sweden), followed by MACS Separators LS column separation (Miltenyi Biotec K.K., Bergisch Gladbach, Germany). Lipids were extracted62 (link) and then dried using N2-gas to produce a dry lipid film in glass tubes. Cholesterol was quantitated using cholesterol oxidase and a coupled fluorescent substrate (Sigma-Aldrich) in a Tecan M200 plate reader. Inorganic phosphates were measured from the total phospholipids recovered using the Malachite Green assay with aliquots of the lipid preparations in chloroform/methanol (1/1, vol/vol). Samples were dried under N2-gas in glass vials, and 0.5 mL of 6 M HCl was added to the vials, which were sealed and incubated for 8 h at 150 °C. The solvent was vacuum-dried, the residue dissolved in water, and then assayed for phosphate content. The samples were also analyzed on an Agilent 0.5 × 150 mm SB C18 capillary column at a flow rate of 12 µL/min, acetonitrile/tetrahydrofuran/water, 65/35/7, gradient to no water, with detection and integration at 215 nm.
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