Protein A beads (KANEKA KanCapA™) were from Wako Chemicals (Osaka, Japan). X-tremeGENE HP was purchased from Sigma (St Louis, MO, USA). Sepasol was purchased from Nacalai Tesque (Kyoto, Japan). The anti-podoplanin antibody (NZ-1, rat IgG) was purchased from Angio Bio Co. (Del Mar, CA, USA) and the anti-CLEC-2 antibody (AF1718, goat IgG) was from Novus Biologicals (Littleton, CO, USA). Horseradish peroxidase (HRP)-labeled anti-human IgG Fab2 antibody (709–1317) was purchased from Rockland Immunochemicals Inc. (Limerick, PA, USA), anti-goat IgG-HRP (P0160) from Dako (Glostrup, Denmark) and anti-Rat IgG-HRP (7077S) from Cell Signaling Tech (Danvers, MA, USA). A set of plasmids for constructing a lentivirus expression vector system, including human immunodeficiency virus type 1 (HIV-1)-based CSII-MCS-Venus, pCAG-HIVgp, and pCMV-VSV-G-RSV-Rev, were obtained from RIKEN (Wako, Osaka, Japan). The Fc-fusion protein expression plasmid containing human IgG1 (pFUSEN-hG1Fc) was purchased from InVIVOGen (San Diego, CA, USA). Hematoporphyrin was from MedChemExpress (Monmouth Junction, NJ, USA). 2CP was synthesized as reported previously [10 (link)].
Sepasol
Manufactured by Nacalai Tesque
Sourced in Japan
Sepasol is a laboratory equipment designed for the separation and purification of chemical substances. It utilizes a centrifugation process to separate components based on their density differences. The core function of Sepasol is to facilitate the efficient isolation and concentration of target analytes from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Thunderbird sybr qpcr mix, by Toyobo (7 mentions)
Revertra ace, by Toyobo (6 mentions)
Lightcycler 96, by Roche (5 mentions)
Revertra ace qpcr rt kit, by Toyobo (4 mentions)
Lightcycler 480, by Roche (3 mentions)
Revertra ace qpcr rt master mix, by Toyobo (3 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Ethanol, by Nacalai Tesque (2 mentions)
2 propanol, by Nacalai Tesque (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Chloroform, by Nacalai Tesque (2 mentions)
Ex taq, by Takara Bio (2 mentions)
Superscript reverse transcriptase, by Roche (2 mentions)
Sybr green, by Toyobo (2 mentions)
Sybr green master mix, by Roche (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Lightcycler system, by Roche (2 mentions)
X tremegene hp, by Merck Group (1 mentions)
Pfusen hg1fc, by InvivoGen (1 mentions)
42 protocols using sepasol
1
Podoplanin and CLEC-2 Interaction Protocol
2
Liver RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from liver tissues using Sepasol (Nacalai Tesque) according to the manufacturer’s protocol. cDNA was generated from total RNA by reverse transcription with Rever Tra Ace (Toyobo, Osaka, Japan). Quantitative reverse transcription (RT)-PCR was performed using StepOne (Applied Biosystems, Waltham, MA, USA) with the Thunderbird SYBR qPCR Kit (Toyobo, Osaka, Japan).The primer sequences are listed in Table 2 .
3
RNA Extraction and qPCR Analysis
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Total RNA was isolated from cultured cells using Sepasol (Nacalai Tesque), and following reverse transcription reaction was performed using a ReverTra Ace qPCR RT kit (TOYOBO). qPCR was performed using THUNDERBIRD SYBR qPCR mix (TOYOBO) in a LightCycler96 (Roche). All reactions were conducted according to the manufacturer’s instructions. The primers are listed in Table S1.
4
RNA Extraction from Zebrafish Embryo Heads
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Heads of 48 hpf wild-type sibling and slbp1 mutant embryos were dissected and transferred to 100 μL Sepasol (Nacalai tesque, 09379) on ice. Heads were then homogenized using a hand homogenizer (~20 pulses). Twenty μL CHCl3 were then added to samples and mixed gently. After centrifugation at 15,000 g for 15 min, the aqueous phase was collected and mixed with 100 μL isopropanol. One μL of RNase-free glycogen (Nacalai tesque 11170–11, 20 mg/mL) was added to all samples to increase the yield. After incubating at room temperature for 10 min, samples were centrifuged at 15,000 g at 4 °C for 15 min. Supernatant was removed and the pellet was washed three times with 500 μL of 75 % ethanol and centrifuged at 8000 g at 4 °C. The pellet was then resuspended in a desired amount of nuclease-free water and stored at –80 °C. RNA concentration and purity of samples were determined using a Nanodrop.
5
Quantitative RT-PCR Analysis of Il31 Expression
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Total RNA was prepared from lungs using Sepasol (Nacalai Tesque, Inc.) and treated with DNase (TURBO DNA-free-kit; Thermo Fisher Scientific Inc., MA). cDNA was synthesized from the isolated RNA by RT-PCR (PrimeScript RT Reagent kit; TAKARA BIO Inc., Shiga, Japan). Quantitative real-time PCR was performed with SYBER Premix Ex Taq (TAKARA BIO Inc.) or SYBER Premix DimerEraser (TAKARA BIO Inc.) using a CFX384TM Touch Real-time PCR Detection System (BioRad Laboratories, Inc., Hercules, CA). Relative gene expression was determined against HPRT gene expression. The following PCR primers were designed: forward primer 5′-ATACAGCTGCCGTGTTTCAG-3′ and reverse primer 5′-AGCCATCTTATCACCCAAGAA-3′ for Il31 mRNA; and forward primer 5′-GGCCAGACTTTGTTGGATTTG-3′ and reverse primer 5′-CGCTCATCTTAGGCTTTGTATTTG-3′ for Hprt mRNA.
6
RNA Isolation and qPCR Analysis
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Total RNA was isolated from cultured cells using Sepasol (Nacalai Tesque), and following reverse transcription reaction was performed using ReverTra Ace qPCR RT kit (TOYOBO). Quantitative PCR (qPCR) was performed using THUNDERBIRD SYBR qPCR mix (TOYOBO) and LightCycler96 (Roche). All reaction was conducted according to the manufacturer’s instruction. Primers are listed in Supplementary Table 2 .
7
Gene Expression Analysis in Skin Tissues
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Isolated skin tissues were homogenized and total RNAs were extracted using Sepasol® (Nacalai Tesque, Kyoto, Japan) and synthesized in a cDNA pool as described previously [20 (link)]. Total RNAs preparation were also performed from six-well culture plates at confluency. Real-time RT-PCR analysis was performed as described previously [20 (link)]. The experiments were performed at five different cDNA pool dilutions. PCR products were normalized against Gapdh and measurements between samples were compared by cycling threshold (Ct). Primer sequences used are summarized in Table S1 . A non-regulated housekeeping gene Gapdh served as an internal control and was used to normalize for differences in input RNA.
8
Gene Expression Analysis of Intestinal Hormones
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Total RNA was extracted using Sepasol (Nacalai Tesque) according to the manufacturer’s instructions. Two micrograms of RNA were used for cDNA synthesis with ReverTra Ace (Toyobo, Tokyo, Japan). The primer sequences used were: 5'-CGTGACATTAAGGAGAAGCT-3' and 5'-CATACTCCTGCTTGCTGATC-3' for ACTB (β-actin), 5'-CGGAAACCTGGAGAACTGCG-3' and 5'-TATCGCAGAGAACGGATGGC-3' for CCK, 5'-CCTTCAAGACACAGAGGAGAAATCC-3' and 5'-CCCCAACCTGTTTACATTTAGCG-3' for GCG (glucagon), 5'-GGGACAGTAGTAGCTTCCCT-3' and 5'-CATTTCCATTCTGGCTGGGA-3' for PC1 (prohormone convertase 1), 5'-TATGGTGTTCGTGCGCAGGC-3' and 5'-GAGGGCCCAGACCTGTGGTGA-3' for PYY. The PCR program was 30 cycles of denaturation at 98°C for 10 s, annealing at 56–58°C for 30 s and extension at 68°C for 30 s. The products were detected on an agarose gel by UV irradiation with ethidium bromide.
9
Quantifying Target Gene Expression with qRT-PCR
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Total ribonucleic acid (RNA) upon treatment with γ-mangostin and α-mangostin was collected from cells using Sepasol (Nacalai Tesque, Japan). The reverse transcription of complementary deoxyribonucleic acid (cDNA) was performed using ReverTra Ace qPCR RT Kit (Toyobo, Japan), which included DNAse I to remove genomic DNA. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) was performed using the Thunderbird SYBR qPCR Mix (Toyobo, Japan) on a Real-Time PCR System Light Cycler 96 (Roche, USA). Rac, Farp, CXCR4 (28 (link)), and latrophilin-2 (LPHN2) primers were used, and GAPDH was detected as the internal control. The primer sequences are listed in Table 1 .
10
Quantitative Analysis of Inflammatory and Antioxidant Genes
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Total RNA was extracted using Sepasol (Nacalai Tesque) according to the manufacturer’s protocol. cDNA was generated using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO) according to the manufacturer’s protocol. Quantification of mRNA was performed with a Light Cycler 480 (Roche) or TP-800 (TAKARA) using THUNDERBIRD SYBR qPCR Mix (TOYOBO). Primer sets used for qRT-PCR analysis were CCL5 forward: 5′-ACCACACCCTGCTGCTTTG-3′, CCL5 reverse: 5′-CACACACTTGGCGGTTCTTTC-3′, SOD2 forward: 5′-TGGAAGCCATCAAACGTGAC-3′, SOD2 reverse: 5′-AAACCAAGCCAACCCCAAC-3′, HO1 forward: 5′-CTTTCAGAAGGGCCAGGTG-3′, HO1 reverse: 5′-GGAAGTAGACAGGGGCGAAG-3′, and actin forward: 5′-TCCCTGGAGAAGAGCTACGAG-3′, actin reverse: 5′-GGAAGGAAGGCTGGAAGAGTG-3′.
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