MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) formazan powder was purchased from Sigma-Aldrich (St. Louis, MO, USA). Petroleum ether and Ethyl acetate were purchased from Global Science (Auckland, NZ), ethanol was purchased from ThermoFisher (Auckland, NZ). Foetal Bovine Serum (FBS) was purchased from Medica Pacifica (Auckland, NZ). Roswell Park Memorial Institute (RPMI) 1640 medium, no phenol red, L- Glutamine (200 mM), Penicillin- Streptomycin (10,000 U/mL), TrypLE™ Express, no phenol red, Trypan blue stain (0.4%), and Dulbecco's Phosphate Buffered Saline (D-PBS) were all purchased from Life Technologies (Auckland, NZ). Dimethyl sulfoxide (DMSO) was purchased from Thermo-Fisher Scientific, (Auckland, NZ); Apo-ONE® Homogeneous Caspase-3/7 Assay kit was purchased from In Vitro Technologies (Auckland, NZ). Alexa Fluor® 488 annexin V/Dead Cell Apoptosis kit was purchased from Thermo-Fisher Scientific (Auckland, NZ).
No phenol red
Manufactured by Thermo Fisher Scientific
Sourced in United States, New Zealand
No phenol red is a laboratory reagent used in cell culture applications. It serves as a pH indicator, providing a visual cue for changes in the acidity or basicity of the culture medium.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
High glucose, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
Stxg wskmx set, by Tokai Hit (2 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Trypan blue stain, by Thermo Fisher Scientific (1 mentions)
Formazan powder, by Merck Group (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Centricon plus 70, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
21 protocols using no phenol red
1
MTT Cytotoxicity Assay Protocol
2
Transient Transfection and Protein Purification
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293-T cells obtained from the American Type Culture Collection (ATCC, Manassas, VA), were grown in T-182 flasks with Dulbeco’s Modified Eagle’s Medium (DMEM) with no phenol red (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 1 μg/ml of amphotericin B. At 70% confluency, the medium was changed to 15 ml DMEM with 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 1 μg/ml of amphotericin B and transfection was carried out using polyethylenimine (PEI, Sigma Inc.) at a 1:4 ratio of DNA:PEI (w/v). pJP008-Ov8FC (40 μg) was diluted in 1.5 ml OPTI-MEM (Gibco) and PEI (160 μl) was added to the diluted DNA. The DNA-PEI mix was incubated for 10 min at room temperature then added dropwise to the cells. After 48 hrs, cell culture supernatant was collected, centrifuged at 18000 xg for 10 min at 4°C and concentrated using Centricon Plus-70 with a 100 kDa cut-off (Merck, Millipore Ltd.) according to the manufacturer’s instructions. Plasmid pJP008-hIgGFC was transfected into 293-T cells and concentrated similarly. Concentrated protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The concentration of Ov8FC was 3.86 mg/ml and hIgGFC was 3.68 mg/ml.
3
Isolation of Dermal Cells from Murine Skin
4
Cryo-CLEM and Cryo-ET of MG-132-Treated HeLa Cells
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HeLa cells (cell line 25) were maintained in a humidified 37 °C incubator with 5% CO2, then cultured in DMEM medium with no phenol red (Gibco), containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. For cryo-CLEM and cryo-ET, cells were plated onto fibronectin-coated 200-mesh gold R2/2 London finder Quantifoil grids (Quantifoil Micro Tools) at a density of 2 × 105 cells/mL. After a 12-h incubation, cultures were treated with MG-132 (1 µg/mL) for 2 to 3 h or left untreated, before being plunge-frozen in liquid ethane/propane mixture using an FEI Vitrobot Mark IV (21 (link)). Immediately before plunge-freezing, 3 µL of a suspension of beads was applied to grids. The bead suspension was made by diluting 500 nm blue (345/435 nm) polystyrene fluorospheres (Phosphorex) with a colloidal solution of 20 nm gold fiducials (Sigma-Aldrich) pretreated with BSA. The gold served as fiducial markers for cryo-tomogram reconstruction, and the blue fluorospheres served as landmarks for registering fluorescence light microscopy (FLM) images from different channels as well as cryo-EM images (24 (link)). Plunge-frozen grids were subsequently loaded into FEI Polara EM cartridges. EM cartridges containing frozen grids were stored in liquid nitrogen and maintained at ≤−150 °C throughout the experiment, including cryo-FLM imaging, cryo-EM imaging, storage, and transfer.
5
Cultivation of Cos-7 cells in vitro
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Cos-7 cells (ATCC CRL-1651) were routinely cultured in Dulbecco’s Modified Eagle Medium, High Glucose, No glutamine, No Phenol Red (Gibco-Fisher Scientific, Ilkirch Graffenstaden, France) supplemented with 1 mM Sodium Pyruvate (Gibco-Fisher Scientific, Ilkirch Graffenstaden, France), Glutamax (Gibco-Fisher Scientific, Ilkirch Graffenstaden, France), Penicillin-Streptomicyn-Glutamin (Gibco-Fisher Scientific, Ilkirch Graffenstaden, France) and 10% Corning Foetal Bovine Serum (Fisher Scientific, Ilkirch Graffenstaden, France). The cells were cultivated in a CO2-enriched atmosphere (5%) at 37 °C.
6
Live-Cell Imaging in Warm Medium
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Cells were seeded in a four-chamber, no. 1.5 glass-bottom dish (Cellvis) and cultured as appropriate for the intended experiment. Before imaging, the medium was replaced with a warm imaging solution [DMEM, high glucose, HEPES, no phenol red (Gibco, 21063029) supplemented with 10% v/v fetal bovine serum]. The dish was immediately transferred to a Tokai Hit (model: STXG-WSKMX-SET) stage-top incubator preheated to 37°C. The dish was allowed to equilibrate for 10 min before initiating acquisition.
7
Assessing Amyloid-beta Cytotoxicity in Cells
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Static experiments were carried out without flow by culturing the cells inside 12-well plates using the same protocol. Cells were incubated in the presence and absence of soluble Aβ25-35 (20 μmol/L) for 24 and 72 h assessing the possible effect of a specific flow rate (50 µl/min, 2 × 10−5 dynes cm−2). After the required incubation time, all the cells in the coverslips were placed in 12-well plates and washed twice with PBS. MTT assay was carried out by adding 1 mg/ml solution of MTT (Sigma M5655) in DMEM/F-12, HEPES, no phenol red from Gibco (catalog number: 11039021) and adding 500 µl of the solution into each of the wells on the coverslips. Cells were incubated for 4 hours at 37 °C protected from light. Then, 350 µl of isopropanol was added to the cells with gentle pipetting up and down and shaking at 400 rpm for 20 mins to dissolve the crystals formed. Finally, the liquid was transferred into a 96 well plate in triplicate for each condition and the optical density (OD) was measured on an IMark absorbance microplate reader (Bio-Rad, UK) with a test wavelength of 570 nm, using isopropanol as blank.
8
Live Cell Imaging Setup Protocol
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Cells were seeded in a four-chamber, no. 1.5 glass-bottom dish (Cellvis) and cultured as appropriate for the intended experiment. Before imaging, the medium was replaced with a warm imaging solution [DMEM, high glucose, HEPES, no phenol red (Gibco, 21063029) supplemented with 10% v/v fetal bovine serum]. The dish was immediately transferred to a Tokai Hit (model: STXG-WSKMX-SET) stage-top incubator preheated to 37 °C. The dish was allowed to equilibrate for 10 min before initiating acquisition.
9
Mesenchymal Stem Cell Culture Protocol
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Hyaluronic acid (40,583) Low molecular weight (MW) 8000–15,000 Da, sodium alginate (A0682), gelatin (G1890), resazurin sodium salt (R7017), 4,6‐Diamidino‐2‐phenylindole (DAPI) (F6057), alizarin red solution, alcian blue 8GX solution and Oil Red O solution were purchased from Sigma‐Aldrich Chemical Co. (St. Louis, MO). 1X Alpha modified Eagle’s minimum essential medium (α-MEM) (AMEM) and L-glutamine was purchased from HyClone (HyClone, Logan, UT). Nonessential amino acids (NEAA), MEM α, nucleosides, no phenol red, Fetal bovine serum (FBS), 1X trypsin TrypLE™ Express 1X, and penicillin–streptomycin are purchased from Gibco (Gibco, CA, USA). Calcein‐AM and propidium iodide (PI) were purchased from Life Technologies Inc. (Carlsbad, CA). Antibodies anti-CD73, CD90, and CD105 were purchased from Merck (Merck KGaA, Darmstadt, Germany), and antibodies anti-phospho-SIRT1 were purchased from Affinity Biosciences (Cincinnati, USA).
10
Directed Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes
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NKX2‐5‐eGFP human ES cells42 (stable reporter line generated from wild‐type HES3 cells 71 ) were cultured in Essential 8™ medium (Gibco) on Matrigel (BD, Corning) without penicillin/streptomycin. Cells were differentiated in a monolayer toward cardiomyocytes as previously described 45 . In short, human ES cells were cultured in E8 until 60% confluent. Cells were then supplemented with 1% DMSO enriched E8 medium for 24 h. On D0, cells were put on BPEL medium supplemented with Activin A (20 ng/ml, R&D Systems), BMP4 (20 ng/ml, R&D Systems), and CHIR99021 (1.5 μM, Axon Medchem). At D3, medium was changed to BPEL with XAV939 (5 μM, Tocris), and on D6, BPEL without any supplements was used. BPEL medium: IMDM, no phenol red (Gibco) and F12 Ham's F12 nutrient Mix (Gibco) in a 1:1 ratio supplemented with 5% (v/v) PFHM‐II (Gibco), 0.25% (w/v) BSA, 1% (v/v) Chemically Defined Lipid Concentrate (Gibco), 0.1% ITS‐X (Gibco), 450 μM α‐MTG (Sigma), 2 mM GlutaMax, 50 μg/ml L‐ascorbic acid 2‐phosphate (Sigma), and 0.25% penicillin/streptomycin (10,000 U/ml, Gibco).
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NKX2‐5‐eGFP human ES cells
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