Endogenous ROS measured by fluorometric assay with specific probes (Ferreira et al., 2013 (link)). Yeast cells from C. gattii and C. neoformans cultured on SDA (37°C for 72 h) were treated with (MIC eugenol = 256 mg/L for C. gattii ATCC 24065, C. neoformans ATCC 28957, and C. neoformans ATCC 62066; 64 mg/L – C. gattii ATCC 32068) and hydrogen peroxide (positive control) for 1 h in RPMI without phenol red (Sigma–Aldrich, St Louis, MO, United States) and incubated with 10 μM of 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Invitrogen, Life Technologies, Carlsbad, CA, United States) for ROS quantification. A growth control was also performed. The fluorescence was measured with a Fluorometer (Synergy 2 SL Luminescence Microplate Reader; Biotek) using excitation and emission wavelengths of 500 nm. The results were expressed as arbitrary units of fluorescence ±SEM. These tests were performed in triplicate.
Rpmi without phenol red
Manufactured by Merck Group
Sourced in Sao Tome and Principe, United States, Germany
RPMI without phenol red is a cell culture medium formulation that is commonly used in laboratory settings. It provides the necessary nutrients and growth factors for the in vitro cultivation of various cell types. The absence of phenol red in this formulation makes it suitable for applications where the presence of this pH indicator could interfere with the experimental procedures or measurements.
Automatically generated - may contain errors
Lab products found in correlation
Sytox green, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
2 7 dichlorofluorescin diacetate dcfh da, by Thermo Fisher Scientific (1 mentions)
Synergy 2 sl luminescence microplate reader, by Agilent Technologies (1 mentions)
Pha l, by Merck Group (1 mentions)
Msc qualified fbs, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Cho ex cell, by Merck Group (1 mentions)
Antibiotic antimycotic, by Merck Group (1 mentions)
Complete rpmi, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
P nitrophenyl n acetyl β d glucosaminide, by Merck Group (1 mentions)
Cellometer auto t4 plus cell counter, by Revvity (1 mentions)
Infinite m200 pro plate reader, by Tecan (1 mentions)
Evos fl, by Thermo Fisher Scientific (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
6 protocols using rpmi without phenol red
1
Measuring Endogenous ROS in Candida
2
Cryopreserved PBMC Proliferation Assay
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Cryopreserved PBMCs were rapidly thawed; washed twice in PBMC media (RPMI without phenol red, Cat # R7509, Sigma Aldrich) containing 10% MSC qualified FBS (cat # 12662011, Thermo-Fisher), 1 mM Glutamax (Cat # 35050061, Thermo Fisher), and 10 mM HEPES, pH 7.4 (Cat # H0887, Sigma Aldrich); and resuspended at 1.5 × 106 PBMCs per mL in PBMC media. In 96-well plate assays (Corning 96-well flat bottomed, Tissue culture treated, polystyrene plates, Sigma Aldrich Cat # CLS3595), 150,000 PBMCs were loaded per well with or without MSC co-culture in a volume of 200 µL. Proliferation was stimulated by the addition of 50 µL of stimulus as indicated in the text. Stimuli included phytohemagglutinin P (PHA-P, Sigma Aldrich Cat # L-1668) and phytohemagglutinin-L (PHA-L, Sigma Aldrich Cat # 431784). After 2, 4, 6, 24, 48, 72, or 96 h incubation at 37°C, 5% CO2, cells in the media were resuspended and aliquots were taken for analysis. In designated experiments, a monocyte-depleted population of PBMCs was obtained by incubating the thawed, washed, resuspended PBMCs at 1.5 × 106 in culture media in a T-75 tissue culture flask overnight. After incubation, monocyte-depleted PBMCs were removed, washed in culture media, and resuspended at 1.5 × 106 cells per mL for use in experiments. After overnight incubation, monocyte-depleted PBMCs were generally 85% viable.
3
Culturing Devil Facial Tumor Cell Lines
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DFT1 cell line C5065 and DFT2 cell line JV were cultured at 35°C with 5% CO2 in cRF10 [10% complete RPMI (Gibco no. 11875-093) with 2 mM l -glutamine, supplemented with 10% heat-inactivated fetal bovine serum, and 1% antibiotic-antimycotic (Thermo Fisher Scientific no. 15240062)]. RPMI without phenol red (Sigma-Aldrich no. R7509) was used to culture FAST protein cell lines when supernatants were collected for downstream flow cytometry assays. Devil peripheral blood cells were cultured in cRF10 at 35°C with 5% CO2. CHO cells were cultured at 37°C in cRF10 during transfections and drug selection but were otherwise cultured at 35°C in cRF5 (5% complete RPMI). For production of purified recombinant proteins, stably transfected CHO cells were cultured in suspension in spinner flasks in chemically defined, serum-free CHO EX-CELL (Sigma-Aldrich no. 14361C) media supplemented with 8 mM l -glutamine, 10 mM Hepes, 50 μM 2-ME, 1% (v/v) antibiotic-antimycotic, and 1 mM sodium pyruvate and without hygromycin.
4
Mast Cell Degranulation Assay
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RBL-2H3 cells (0.3 × 106) adhered on plate were washed with RPMI (without phenol red) (Sigma, MO, USA) and co-cultured with L. donovani and L. tropica in a final volume of 1.5 ml. Plates were incubated at 37 °C, aliquots of the medium were withdrawn at various times and β-hexosaminidase activity released into the medium was measured. Mock degranulation studies were carried out in parallel by using medium alone59 (link). To determine β-hexosaminidase activity, aliquots of the supernatants and cell lysates were incubated with the substrate solution ((Sigma, MO, USA) 1.3 mg/ml of p-nitrophenyl- N-acetyl-β-D-glucosaminide in 0.1 M citrate buffer (pH 4.5) for 90 mins at 37 °C. Absorbance was read at 405 nm and the amount of exocytosis was expressed as the percentage of total β-hexosaminidase activity released in the supernatant.
5
Measuring Intracellular Oxidative Stress
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Measurement of intracellular DCFDA fluorescence was performed using the Abcam DCFDA Cellular Reactive Oxygen Species Detection Assay Kit (Abcam, Cambridge, MA, ab113851) according to the manufacturer’s instructions. Briefly, 25,000 A549 cells were plated in each well of a 96 well plate in 200 µL RPMI without phenol red (Sigma Aldrich). Cells were allowed to attach for 8 hr. Subsequently, the media on the cells was changed to 200 µL fresh media containing DMSO, CB-839 and NAC as indicated. After 24 hr of treatment, the cells were washed twice with 200 µL of 1x Buffer included in the assay kit. Cells were then incubated with 100 µL of 1x Buffer containing 10 µM DCFDA for 45 min. at 37°C. An unstained control was included, and incubated with 100 µL of 1x Buffer alone. After staining, the DCFDA containing solution was removed and cells were resuspended 100 µL of PBS. DCFDA fluorescence was measured using an Infinite M200Pro plate reader (Tecan) in fluorescence mode with excitation at 485 and emission at 535. Cells were then detached with trypsin-EDTA solution and counted using a Cellometer Auto T4 Plus Cell Counter (Nexcelom Bioscience). The ratio of DCFDA signal intensity to cell number was subsequently computed.
6
Neutrophil Extracellular Trap Formation
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Isolated neutrophils were prepared at 2 × 105 cells/mL in RPMI without phenol red (Sigma-Aldrich, Darmstadt, Germany). Sytox Green (Thermo Fisher Scientific, Waltham, MA, USA) was added in a concentration of 1 μM. After exposure to ELF-PEMF, cells were immediately stimulated with 100 nM PMA, 25 µg/mL LPS, or 0.003% H2O2. For the normalization of the extracellular DNA, neutrophils were lysed with 1% Triton X-100 solution. The cells were incubated at 37 °C with 5% CO2 and the fluorescent intensity was measured at Ex 485 nm/Em 520 nm with a microplate reader every 30 min (FluoStar Omega, BMG Labtech, Ortenberg, Germany). Imaging was performed using a fluorescent microscope (EVOS FL, Life Technologies, Darmstadt, Germany) after 3 h of incubation.
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