C-PC was quantified by UV-Visible electronic absorption using an external calibration method [4 (link)]. Briefly, one hundred milligrams of the lyophilized samples were ground with seasand and extracted by four times sonication for 30 min each with 30 mL of phosphate buffer saline (PBS) (pH 7.0) buffer solution. The extract was centrifuged at 20,000× g at 4 °C for 6 h and the supernatant was filtered through a 0.45 μm membrane filter (Whatman, PTFE, 13 mm). Two hundred microliters of each extract was transferred to a 96 well plate, where a microplate reader (BioTek, Synergy H1, Winooski, VT, USA) was used to detect the absorbance at 620 nm. Working calibration solutions in the range of 0.5–5 mg/mL were prepared by diluting the stock solution of C-phycocyanin (Sigma, St. Louis, MO, USA) with PBS buffer. All of the procedures were performed under subdued light to avoid pigment degradation as described above.
C phycocyanin
Manufactured by Merck Group
Sourced in United States
C-phycocyanin is a natural pigment protein derived from the cyanobacterium Spirulina. It is a light-harvesting chromoprotein with a vibrant blue color. The core function of C-phycocyanin is to absorb specific wavelengths of visible light to facilitate photosynthesis in cyanobacteria.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Whatman, by Cytiva (1 mentions)
Triton x 100, by Merck Group (1 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Jc 1 dye, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Anhydrous ethanol, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
H2so4, by Merck Group (1 mentions)
Nano3, by Merck Group (1 mentions)
Kmno4, by Merck Group (1 mentions)
8 protocols using c phycocyanin
1
Quantification of C-Phycocyanin by UV-Vis Spectroscopy
2
Evaluating Oxalate and C-Phycocyanin Effects
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Oxalate, C-phycocyanin (CP), DMEM (Dulbecco's Modified Eagle Medium), fetal bovine serum (FBS), propidium iodide (PI), trypan blue, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), dimethyl sulfoxide, trypsin, Tween 20, and Triton X-100 were purchased from Sigma Chemicals (St. Louis, MO, USA). 2, 7–dichlorofluorescein diacetate (DCF-DA) (Molecular probes, Inc. USA), and JC-1 dye (Molecular probes, Inc. USA) were procured from Molecular Probes, Inc. (USA), P-JNK/SAPK and P- ERK1/2 antibodies from Cell signaling Technology, USA. T-JNK and T-ERK1/2 antibodies were purchased from Santa Cruz Biotechnology, INC, USA.
3
Synthesis and Characterization of Graphene Oxide
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Penicillin–streptomycin, trypsin–ethylenediaminetetraacetic acid (EDTA), Dulbecco’s Modified Eagle’s Medium, Roswell Park Memorial Institute 1640 medium, and 1% antibiotic–antimycotic were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Cis, fetal bovine serum and an in vitro toxicology assay kit were purchased from Sigma-Aldrich (St Louis, MO, USA). C-phycocyanin, silver nitrate, graphite (Gt) powder, NaOH, KMnO4, NaNO3, anhydrous ethanol, 98% H2SO4, 36% HCl, 30% H2O2 aqueous solution, and all other chemicals were purchased from Sigma-Aldrich unless otherwise stated.
4
Identification of Phycocyanin in Spirulina maxima
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To identify the phycocyanin from water extracted S. maxima sample, extracted S. maxima and purchased C-phycocyanin were analyzed by HPLC-DAD. C-phycocyanin from Spirulina sp. was purchased from Sigma–Aldrich (St. Louis, MO, USA, Cat # P2172). The high-performance liquid chromatography (HPLC) (Dionex) equipped with an LPG 3 × 00 pump, an ACC-3000 autosampler, a DAD-3000 (RS) diode array ultraviolet (UV)/visible detector, and a column oven. Both samples were separated on a Jupiter C5 column (5 μm, 300 Å, 4.6 mm × 250 mm) at 25°C. The mobile phase consisted of 20% (v/v) aqueous acetonitrile (ACN) solution containing 0.1% (v/v) trifluoroacetic acid (TFA) and all reagents were purchased from J. T. Baker as HPLC grade. The flow rate was 1.0 mL/min and the concentration of S. maxima sample was 100 ppm and phycocyanin was 1000 ppm. The injection volume of each sample was 20 μL. The output signal of the detector was recorded using a Dionex Chromeleon™ Chromatography Data System. The UV wavelength was 580 nm and 640 nm, respectively, and the chromatograms were acquired at 580 nm.
5
Seawater Composition for Cell Culture
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C-phycocyanin (30–50% protein content) and lipopolysaccharide (LPS, compound from Pseudomonas aeruginosa) were purchased from Sigma (San Diego, MO, USA). Cell culture reagents, including Dulbecco’s Modified Eagle Medium, fetal bovine serum (FBS), Penicillin-Streptomycin, and Trypsin-EDTA (0.25%), were purchased from Gibco Life Technologies (Waltham, MA, USA). Antibodies against F4/80, NLRP3, PYCARD (ASC), Caspase-1, cleaved-Caspase-1, iNOS, NF-κB p65, p-NF-κB p65, IκBα, and p-IκBα were obtained from Affinity (Affinity Biosciences LTD, USA). In contrast, α-tubulin, horseradish peroxidase (HRP)-labeled secondary antibodies, and CoraLite488 and 555-conjugated secondary antibodies were purchased from Proteintech Group (Chicago, USA).
The experimental seawater was prepared according to the formula provided by the Third Institute of Oceanography, Ministry of Natural Resources, Fujian, China. The main components and contents of the seawater were close to those of the southeast coastal seawater of China, pH 8.2, specific gravity 1.05, and osmotic pressure 1300 mmol/L. The SW components were as follows: NaCl 26.518 g/L, MgCl2 2.447 g/L, MgSO4 3.305 g/L, CaCl2 1.141 g/L, NaBr 0.083 g/L, NaHCO3 0.202 g/L, KCl 0.725 g/L.
The experimental seawater was prepared according to the formula provided by the Third Institute of Oceanography, Ministry of Natural Resources, Fujian, China. The main components and contents of the seawater were close to those of the southeast coastal seawater of China, pH 8.2, specific gravity 1.05, and osmotic pressure 1300 mmol/L. The SW components were as follows: NaCl 26.518 g/L, MgCl2 2.447 g/L, MgSO4 3.305 g/L, CaCl2 1.141 g/L, NaBr 0.083 g/L, NaHCO3 0.202 g/L, KCl 0.725 g/L.
6
Cytoprotective Effects of Spirulina and C-phycocyanin
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The rat pancreatic β-cell line RINm5F was purchased from the American Type Culture Collection and cultured in a humidified incubator at 37 °C with 5% CO2. The cells were grown in RPMI-1640 medium supplemented with 2500 mM glucose, 10 mM HEPES, 18 mM sodium bicarbonate, 1 mM sodium pyruvate (Sigma, St. Louis, MO, USA), 10% fetal bovine serum, 100 U/mL potassium penicillin, and 100 mg/mL streptomycin sulfate (Gibco, Grand Island, NY, USA).
For treatment, lyophilized Spirulina extract or C-phycocyanin (PC; Sigma) was dissolved in the culture medium. Lipopolysaccharide of the Spirulina extract was removed by incubation with polymixin B (Sigma) as previously described [38 ]. Cells were co-treated with or without IL-1β (50 U/mL), IFN-γ (100 U/mL) (R&D Systems, Abingdon, UK), Spirulina extract (1 μg/mL), and PC (1 μg/mL) to evaluate the protective effects of Spirulina extracts. PC is a phycobiliprotein with nutritional and therapeutic value showing antioxidant, anti-inflammatory, neuroprotective, and hepatoprotective effects. Since PC is a known constituent of Spirulina [39 (link)], it was used to directly compare the effects to those of Spirulina extract.
For treatment, lyophilized Spirulina extract or C-phycocyanin (PC; Sigma) was dissolved in the culture medium. Lipopolysaccharide of the Spirulina extract was removed by incubation with polymixin B (Sigma) as previously described [38 ]. Cells were co-treated with or without IL-1β (50 U/mL), IFN-γ (100 U/mL) (R&D Systems, Abingdon, UK), Spirulina extract (1 μg/mL), and PC (1 μg/mL) to evaluate the protective effects of Spirulina extracts. PC is a phycobiliprotein with nutritional and therapeutic value showing antioxidant, anti-inflammatory, neuroprotective, and hepatoprotective effects. Since PC is a known constituent of Spirulina [39 (link)], it was used to directly compare the effects to those of Spirulina extract.
7
Cytotoxicity and Apoptosis Assay Protocol
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C-phycocyanin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and staining components, such as acridine orange, ethidium bromide, Hoechst 33342, rhodamine-123 (Rho-123), 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA), 1,3-diphenylisobenzofuran (DPBF), and Annexin V-Cy3 apoptosis detection kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Singlet oxygen sensor green (SOSG) was obtained from Thermofisher Scientific (Waltham, MA, USA). All of the chemicals purchased were used as received without further purification.
8
Spirulina Cultivation and Seed Sourcing
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Dried biomass Spirulina platensis was obtained from the Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang (KMITL). Algal cultures were grown in Zarrouk medium [13] and maintained in a 0.03% CO2 atmosphere at 25 o C and pH 10.5. The cultivation flasks were illuminated under 400 µmolm -2 s -1 light intensity. Cells were harvested at the late exponential phase by centrifugation and dried in an oven at 40 o C. Analytical grade C-phycocyanin was purchased from Sigma-Aldrich, (Saint Louis, MO, USA). All other chemicals used were of reagent grade. The seeds for barnyardgrass (Echinochloa crus-galli (L.) Beauv.) were randomly collected from rice fields in Phisanulok Province, Thailand in August 2018, and Chinese amaranth (Amaranthus tricolor L.) was purchased from Thai Seed & Agriculture Co., Ltd. company, Bangkok, Thailand. The germination rate of the test seeds was > 80%.
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