Cells were incubated with 5-aminolevulinic acid (5-ALA) (Sigma Aldrich, Munich, Germany) dissolved in culture media to achieve 3 mM concentration. After 4 h of incubation, cells were rinsed twice with phosphate buffered saline (PBS). Irradiation was carried out in cell culture media without FBS and phenol red. Cells were irradiated with red light using a halogen lamp (Penta Lamps, Teclas, Lugano, Switzerland). Five joules per centimeter squared of energy was delivered during 83 s exposure with 50 mW/cm2 to each sample at the wavelength 630+/−20 nm. Control groups comprised of cells exposed to light only, 5-ALA, or left without any treatment. After cells irradiation, thalidomide (Sigma Aldrich) was added to selected wells. We used 20 mg/mL stock concentration of thalidomide, which was dissolved in DMSO, then the appropriate volume was added to culture media to obtain 20 µg/mL final concentration. DMSO concentration in media did not exceed 0,5% and did not affect the cells. Cells were incubated with thalidomide for 48 h. The second control group of cells comprised those exposed to TMD for 48 h only.
5 aminolevulinic acid 5 ala
Manufactured by Merck Group
Sourced in Germany, United States, China
5-aminolevulinic acid (5-ALA) is a chemical compound used in laboratory settings. It functions as a precursor in the biosynthesis of important biomolecules, including heme and chlorophyll. 5-ALA serves as a valuable tool for researchers studying these biological processes.
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Lab products found in correlation
Thalidomide, by Merck Group (1 mentions)
Sodium citrate trihydrate, by Merck Group (1 mentions)
Cobalt chloride hexahydrate, by Merck Group (1 mentions)
Gold 3 chloride solution, by Merck Group (1 mentions)
Polyvinylpyrrolidone pvp, by Merck Group (1 mentions)
Protoporphyrin 9 ppix, by Merck Group (1 mentions)
Sodium borohydride, by Merck Group (1 mentions)
Multimode microplate reader, by Agilent Technologies (1 mentions)
5 ala, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Dichloroacetic acid dca, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mcf 7 cell line, by American Type Culture Collection (1 mentions)
Penicillin, by American Type Culture Collection (1 mentions)
Streptomycin, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Dmem media, by American Type Culture Collection (1 mentions)
Methionine, by Merck Group (1 mentions)
Ferrous ammonium sulfate, by Merck Group (1 mentions)
Isoleucine, by Merck Group (1 mentions)
9 protocols using 5 aminolevulinic acid 5 ala
1
Red Light-Activated Photodynamic Therapy
2
Cobalt-Gold Nanoparticle Synthesis
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Cobalt chloride hexahydrate (CoCl2•6H2O 99.99%), sodium borohydride (NaBH4 99%), sodium citrate trihydrate, gold(III) chloride solution (30 wt% of HAuCl4 in dilute HCl), poly(vinylpyrrolidone) (PVP) (weight-average molecular weight=2,500 g/mol), 5-aminolevulinic acid (5-ALA), and protoporphyrin IX (PpIX) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The deionized water used in this study was 18.2 MΩ Milli-Q filtered by Quantum Ex, Ultrapure Oranex Cartridge filtration columns (EMD Millipore, Billerica, MA, USA).
3
CYP2C8 Mutant Library Screening
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The constructed random mutant libraries of CYP2C8 were transformed into E. coli DH5α cells. The colonies on agar plates with LB media were inoculated into each well of the 96-well tissue culture test plates filled with 200 μL of TB media, including ampicillin (50 μg/mL), 0.5 mM 5-aminolevulinic acid (5-ALA, Sigma-Aldrich), 1.0 mM isopropyl β-d -thiogalactoside (IPTG), 1.0 mM thiamine, and trace elements. Approximately 300 colonies were picked for each round of screening. Randomly mutated CYP2C8 enzymes were expressed at 28 °C for 48 h, and then the culture media were removed by centrifugation. The luminescent activity of the mutants was measured by adding a 2× reaction mixture (M9 minimal medium (including 1% glucose, w/v) and luciferin-ME (300 μM)) to each well of the 96-well plates. The reaction was stopped by adding an LDR (including luciferase, P450-GloTM Assays) after incubation at 37 °C for 4 h, and luminescence was detected using a Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA).
4
3D Engineered Stem Cell Tumor Invasion
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3D bioengineered constructs with healthy differentiated induced neural stem cells (hiNSCs) were generated using a similar methodology as described for tumor cultures and previously detailed for hiNSCs in the silk-scaffold system66 (link), with further optimization. The differentiating hiNSC constructs were transfected with a viral vector, AAVdj-hSyn-eYFP (Charu Ramakrishnan, Karl Deisseroth, Stanford University), such that the differentiated synapsin (Syn)-expressing neurons would express eYFP (Yellow fluorescent protein). GBM cells expanded as spheres from the patient-tumor were placed in close proximity to the 3D hiNSC constructs 7 mo post-differentiation. Before starting the co-cultures, the GBM cells were separately incubated with 5-aminolevulinic acid (5-ALA, Sigma–Aldrich A3785) for 48 h, for visualization following 5-ALA preferential accumulation. The invasion of healthy hiNSC cultures by the GBM cells and vice versa was observed 48 h post co-culture, by simultaneous live imaging of the differentiated hiNSCs by eYFP and GBM by 5-ALA.
5
Breast Cancer Cell Line MCF-7 Assay Protocol
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Human breast adenocarcinoma MCF-7 cell line, DMEM media, Fetal Bovine Serum (FBS), penicillin, streptomycin and other cell culture reagents were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Vybrant flow cytometry apoptosis assay kit (Catalog # V-13243) was obtained from Molecular Probes (Carlbad, CA). Dichloroacetic acid (DCA), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, dimethylsulfoxide (DMSO), 5’,5’,6’,6’-tetrachloro-1’,1’,3’,3’-iodide (JC-1) dye, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence dye, Trypan blue, 5-aminolevulinic acid (5-ALA) and ATP assay kit (Colorimetric/ Fluorometric, Cat. ab83355) were obtained from Sigma-Aldrich (St. Louis, MO).
6
Comprehensive Biochemical Reagent Inventory
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Ammonium acetate, acetonitrile, phenylalanine, glycerol, and d -glucose were purchased from Fisher Scientific. Arabinose, 5-aminolevulinic acid (5-ALA), ascorbic acid, biotin, bovine liver catalase, chloramphenicol, citraconic acid, copper (II) sulfate, 5′deoxyadenosine (5 dA), 5′-deoxy-5’-(methylthio)adenosine (MTA), diethylamine NONOate, diethylenetriamine pentaacetic acid (DTPA), dithiothreitol (DTT), ferrous ammonium sulfate (FAS), horseradish peroxidase, hydrogen peroxide, imidazole, isopropyl β-d -1-thiogalactopyranoside (IPTG), lipoic acid, malic acid, potassium ferricyanide, S-adenosylmethionine (SAM), silver nitrate, superoxide dismutase (SOD), sodium dithionite, xanthine, xanthine oxidase, histidine, cystine, isoleucine, methionine, tyrosine, tryptophan, and valine were obtained from Sigma. Amicon centrifugal filters were from Millipore, and Amplex Red was from Invitrogen. His GraviTrap™ was purchased from GE Healthcare.
7
5-ALA Photodynamic Therapy for CRC
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5-aminolevulinic acid (5-ALA) (Sigma-Aldrich, Shanghai, China) was used as photosensitizers in this study. CRC cells were incubated with 0, 5, 10, or 20 mg/L 5-ALA for 6 h and then subjected to PDT experiments. Photosensitized cells were briefly irradiated with visible light from LD-630 laser tumor therapeutic equipment (Leimai Tech, Shenzhen, China). The average fluence rate was 15 mW/cm2. A light dose of 60 J/cm2 was used. The PDT-treated or non-treated CRC cells were then examined for cell viability 24 h after photodynamic therapy, cell apoptosis, NEAT1 expression levels, and so on, were examined as well.
8
Media Preparation and Supplementation
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All strains, media, and growth conditions used in this study are listed in Table 1. Lysogeny broth (LB, HiMedia), nutrient broth (NB; HiMedia), Reasoner's 2A media (R2A, HiMedia), and Reasoner's 2A agar (R2A agar, HiMedia) were produced according to manufacturer's instructions. Media were supplemented with 1.5% bacteriological agar (Oxoid) where needed. To support growth of E. coli ST18, media was supplemented with 50 mg L -1 5-aminolevulinic acid (5-ala, Sigma). When appropriate, media were supplemented with antibiotics with the following concentrations: 100 mg L -1 ampicillin, 20 mg L -1 chloramphenicol, 10 mg L -1 colistin, 100 mg L -1 erythromycin, 15 mg L -1 gentamicin, and/ or 50 mg L -1 kanamycin.
9
Necroptosis Signaling Pathway Modulation
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5-Aminolevulinic acid (5-ALA) and p38 inhibitor (S8307) was purchased from Sigma-Aldrich. MK2 inhibitor (MK2i, 475864) was bought from Merck Millipore. TNF-α was from Peprotech and zVAD-fmk was purchased from Promega. The BV6 Smac mimetic was previously described [21] . The following antibodies were used for western blot analysis: anti-FLAG-M2 (F-3165) and anti-RIP3 rat (R4277) (Sigma-Aldrich); anti-Caspase-8 (ALX-804-429) and anti-LC3 (NT-0231-100) (Enzo Life Sciences); anti-RIP1 (3493), anti-RIP3 human (13526), anti-TSC2 (4308), anti-Phospho-TSC2 rat (ser 1254) (3611), Phospho-4E-BP1 (Thr37/46) (236B4) and anti-Phospho-p38 MAPK (Thr180/Tyr182) (9211) (Cell Signalling Technology), anti-ATG7 (sc-12497), anti-YWHAE (sc-23957), anti-YWHAZ (sc-1019), anti-MK2 (sc-6221), anti-HA probe (sc-7392) (Santa Cruz Biotechnology); anti-Tuberin phospho S1254 human (ab133454) (Abcam); anti-GAPDH (AM4300) (Ambion).
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