The quantitative analysis of aggrecan and collagen II genes was adopted to evaluate the NP-like cells which differentiated from rBMSCs. RBMSCs were incubated in 48 well plates for 1, 2, and 4 weeks, after which the cell proliferation was determined by real-time qRT-PCR assays. Three samples were taken from each group for evaluation, and acellular scaffolds were used as the blank control group. The RNA of NP-like cells was extracted by a MiniBEST Universal RNA Extraction Kit (Takera, Japan). Real-time polymerase-chain reaction (PCR) was performed using SYBR® Green PCR Master Mix (Takera, Japan), and glyceraldehyde-3-phosphate dehydrogenase (Invitrogen, USA) was used as the endogenous control. The RNA data were analyzed by ABI PRISM® 7500 Sequence Detection System. Data were presented as mean with standard deviation.
Glyceraldehyde 3 phosphate dehydrogenase
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
Glyceraldehyde 3-phosphate dehydrogenase is an enzyme involved in the glycolytic pathway. It catalyzes the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (2 mentions)
Rnalater, by Merck Group (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Trisure reagent, by Meridian Bioscience (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Proteinase inhibitor cocktail, by Roche (1 mentions)
Anti phospho stat1, by Cell Signaling Technology (1 mentions)
Ionomycin, by Merck Group (1 mentions)
High capacity cdna reverse transcription reagents, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Iblot transfer system, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Dylight 800, by Thermo Fisher Scientific (1 mentions)
23 protocols using glyceraldehyde 3 phosphate dehydrogenase
1
Quantitative Analysis of NP-like Cells
2
Quantifying Mitochondrial Protein Expression
3
RNA Isolation and cDNA Synthesis
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Tissues from euthanized mice at end-point or freshly isolated hPBMCs were stored in RNAlater (Sigma-Aldrich) at -20°C. RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) as per the manufacturer's instructions. Isolated RNA was immediately converted to complementary DNA (cDNA), using the qScript cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA, USA) as per the manufacturer's instructions, and RNA stored at -80°C. cDNA was checked by PCR amplification of the house keeping gene glyceraldehyde 3-phosphate dehydrogenase (Invitrogen, Carlsbad, CA, USA) for 35 cycles (95°C for 1 min, 55°C for 1 min, and 72°C for 1 min) and a holding temperature of 4°C. Purity and size of amplicons were confirmed by 2% agarose gel electrophoresis.
4
RNA Isolation and cDNA Synthesis
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Tissues removed from euthanized mice were stored in RNAlater (Sigma-Aldrich) at -20°C until required. RNA was isolated using TRIsure reagent (Bioline, London, UK), as per the manufacturer's instructions. Isolated RNA was converted immediately to cDNA using the qScript cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA, USA), as per the manufacturer's instructions, and stored at -20°C. cDNA was checked by polymerase chain reaction (PCR) amplification of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (Invitrogen, Carlsbad, CA, USA) for 35 cycles (95°C for 1 min, 55°C for 1 min and 72°C for 1 min) and a holding temperature of 48C. Purity and size of amplicons were confirmed by 2% agarose gel electrophoresis.
5
Quantification of Protein Levels in Nasal Tissue and Macrophages
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Total protein of nasal tissue sections from the noninfection and infection models and THP1-differentiated macrophages with or without S aureus infection was extracted by using RAPI buffer (Invitrogen) with cocktail proteinase inhibitor (Roche, Mannheim, Germany). The following antibodies were used for Western blotting: anti-phospho-STAT1 (1:1000) and anti-STAT1 (1:1000) were purchased from Cell Signaling Technology (Danvers, Mass), and anti-IL-28R antibody (1:800, sc-135101) was purchased from Santa Cruz Biotechnology. Relative band densities of the target protein to glyceraldehyde-3-phosphate dehydrogenase (Invitrogen) were estimated with Lab Image Analysis Software (Bio-Rad Laboratories).
6
Analyzing Kidney Tissue Compartments
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Unfixed biopsy cores were exposed to RNase inhibitor and microdissected into glomerular (including Bowman’s capsule) and tubular specimens. RNA was isolated from each kidney tissue compartment with an RNeasy Micro kit (Qiagen). Subsequently, RNA was converted to cDNA using a kit according to the manufacturer’s protocol (Promega Biotech). The TaqMan gene expression assay was used for human BAFF, APRIL, synaptopodin, and Wilms’ tumor 1, with the endogenous control (glyceraldehyde 3-phosphate dehydrogenase) (Thermo Fisher Scientific). The quantitation of the results was performed by the comparative Ct (2-(delta)(delta)Ct) method.
7
CD40L Expression Profiling by Flow Cytometry
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CD40L protein expression was determined by flow cytometry as previously described [14 (link), 26 (link)]. Platelets were obtained and assessed by flow cytometry for CD40L protein expression before and after stimulation with 0.2 U/ml thrombin for 5 minutes at 37 °C in a 5% CO2 atmosphere [14 (link)]. CD40L protein expression in monocytes and in CD4+ and CD8+ T lymphocytes was determined by flow cytometry before and after stimulation with 1.5 μM ionomycin (Sigma-Aldrich) and/or 25 ng/ml PMA [26 (link)]. CD40L gene expression in PBMCs was determined before and after stimulation with 10 ng/ml PMA for 3 h using the TaqMan® Gene Expression qRT-PCR Assay for CD40L (Thermo Fisher Scientific). cDNA levels were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (Thermo Fisher Scientific).
8
Quantifying Stem Cell Markers Expression
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RNA from paired MSC samples was extracted using the RNeasy mini kit (Qiagen, Germany) as per the manufacturer’s protocol. RNA purity and quantity was analysed using Nanodrop 2000 (Thermo Fisher Scientific, USA) and reverse transcribed using High-Capacity cDNA reverse transcription reagents (Applied Biosystems, USA). Taqman® (Thermo Fisher Scientific, USA) gene expression assays were used to measure the expression levels of the genes CDCP1 (CD318, Hs01080405_m1), ALPL (MSCA-1, Hs01029144_m1) and DPP4 (CD26, Hs00897391_m1) all purchased from Thermo Fisher Scientific, USA. Reactions were carried out in triplicate using 7900HT Fast Real Time PCR system (Applied Biosystems, USA) and standard thermal cycling conditions. Ct values were normalised to the reference gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs9999905_m1, Thermo Fisher Scientific, USA).
9
Protein Extraction and Western Blotting
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Proteins for MDCK cells were extracted using lysis buffer [100 mM tris (pH 7.5) + 150 mM NaCl + 0.5% NP-40 + 10% glycerol + 0.5% Triton X-100] containing 1× protease inhibitor cocktail (Roche) and 1× phosphatase inhibitor (PhosphosSTOP, Roche). Protein (20 μg) was loaded onto NuPAGE 4 to 12% bis-tris gel using a mini gel tank and dry-transferred using an iBlot transfer system (Invitrogen). Nonspecific sites were blocked using 5% nonfat dry milk in 0.1% phosphate-buffered saline (PBS) with Tween 20. Primary antibodies (glyceraldehyde-3-phosphate dehydrogenase from Thermo Fisher Scientific, reference mA5-15738, or cadherin-6 from Cell Signaling Technology, reference 48111) were diluted in PBS with Tween 20 at 1:1000, and the blots were incubated overnight on a shaker at 4°C. The blots were then washed three to four times for 10 min each in PBS with 0.1% Tween 20 and incubated with either horseradish peroxidase–linked (Pierce) or Dylight 800 (Thermo Fisher Scientific)–linked secondary antibodies at 1:10,000 for 2 hours. The blots were then washed three times PBS with 0.1% Tween 20 or tris-buffered saline with Tween 20 for 10 min each. The blots were then revealed using ChemiDoc MP (Bio-Rad) using SuperSignal West Femto (Thermo Fisher Scientific, 34095) or chemiluminescence.
10
Immunoblotting of Autophagy and Inflammatory Markers
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After co-culture with LPS/ATP, rapamycin and/or bafilomycin or EPEC (MOI = 5), unstimulated/uninfected and stimulated/infected BMDCs (WT/WAS KO) and THP-1 (WT and WAS KO) were lysed, and whole-cell extracts were quantitated by the Bradford assay (Bio-Rad). Supernatant proteins were precipitated overnight in 80% ice-cold acetone. Equal amounts of cell lysates or supernatants were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and were transferred to nitrocellulose or polyvinylidene fluoride membrane (GE Healthcare, Buckinghamshire, UK). The membranes then were incubated with polyclonal rabbit anti-mouse IL-1β (1:500, H153; Santa Cruz Biotechnology), monoclonal anti-mouse caspase-1 (1:1,000, Casper-1; Adipogen), polyclonal rabbit anti-mouse LC3 (1:1,000, PM036; MBL International) or polyclonal rabbit anti-mouse SEPT2 (1:1,000, 11397-1-AP; Proteintech), followed by an horseradish peroxidase-conjugated secondary antibody. Membranes were stripped and re-probed for β-actin (1:20,000; Sigma) or glyceraldehyde 3-phosphate dehydrogenase (1:20,000; Thermo Fisher Scientific) as a loading control. The immunoreactive bands were detected using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare).
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