Mitochondria from rhesus monkey skeletal muscle (5, 20 or 50 mg) was isolated using the Mitochondria Isolation Kit from Abcam (Cambridge, UK), with slight modifications. Briefly, the skeletal muscle was suspended in 2.5 ml of Reagent A. After incubation on ice for 10 min, the skeletal muscle was homogenized for 30 seconds with a PRO200 homogenizer (PRO Scientific, Oxford, CT, USA). The resulting suspension was centrifuged for 10 min at 1000 × g and 4 °C. The supernatant was saved (no. 1) and the pellet was resuspended in 2.5 ml of Reagent B. Homogenization and centrifugation steps were repeated and the resulting supernatant was saved (no. 2). Supernatants 1 and 2 were mixed thoroughly and centrifuged for 15 min at 12,000 × g and 4 °C to pellet the mitochondria.
Pro200
Manufactured by PRO Scientific
Sourced in United States
The PRO200 is a precision digital scale designed for laboratory use. It features a sturdy stainless steel platform and can accurately measure weights up to 200 grams with a resolution of 0.01 grams.
Automatically generated - may contain errors
Lab products found in correlation
Rna 6000 nano labchip kit, by Agilent Technologies (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mitochondria isolation kit, by Abcam (1 mentions)
Middle brook 7h11 plate, by BD (1 mentions)
Nanodrop nd 100 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Abi 7500, by Thermo Fisher Scientific (1 mentions)
Trizol rna extraction, by Thermo Fisher Scientific (1 mentions)
Abi high capacity cdna rt kit, by Thermo Fisher Scientific (1 mentions)
Qubit 3 fluorometer, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Chloramphenicol, by Roche (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Ypd agar, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Evolution 220 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
28 protocols using pro200
1
Rhesus Monkey Skeletal Muscle Mitochondria Isolation
2
Tissue Distribution of Paclitaxel Formulations
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Tissue-distribution studies were performed to measure the concentrations of PTX quantitatively in different organs of tumor-bearing nude mice. BEL7402 cancer cells (107) were inoculated into male BALB/c nude mice (6–8 weeks, 20±2 g). When tumor volumes had reached ~200 mm3, the mice were randomly divided into three groups (n=5) and treated with Taxol, PTX-Ms, and PTX-CMs at 10 mg PTX/kg. Mice were killed at 2 hours posttreatment to harvest hearts, livers, spleens, lungs, kidneys, and tumors. Tissue samples were snap-frozen in liquid N2 and stored at −80°C until further analysis.
Tissue concentrations of PTX were determined by HPLC as described previously.34 (link) Briefly, tissue samples were homogenized with a PRO200 (PRO Scientific, Oxford, CT, USA) with physiological saline at a ratio of 1:2 g/mL. Tissue homogenate samples (200 μL) were mixed with 50 μL internal standard diazepam (10 μg/mL in acetonitrile) by vortex mixing for 30 seconds. Then, samples were extracted with 750 μL acetonitrile using vortex shaking for 3 minutes and centrifuged at 15,000 g for 10 minutes. The upper organic layer was transferred to autosampler vials and analyzed by HPLC. Standard samples were prepared in duplicate from 0.05 to 50 μg/mL (0.05, 0.1, 1, 5, 25, 50 μg/mL). Analysis conditions were same as the in pharmacokinetic evaluation.
Tissue concentrations of PTX were determined by HPLC as described previously.34 (link) Briefly, tissue samples were homogenized with a PRO200 (PRO Scientific, Oxford, CT, USA) with physiological saline at a ratio of 1:2 g/mL. Tissue homogenate samples (200 μL) were mixed with 50 μL internal standard diazepam (10 μg/mL in acetonitrile) by vortex mixing for 30 seconds. Then, samples were extracted with 750 μL acetonitrile using vortex shaking for 3 minutes and centrifuged at 15,000 g for 10 minutes. The upper organic layer was transferred to autosampler vials and analyzed by HPLC. Standard samples were prepared in duplicate from 0.05 to 50 μg/mL (0.05, 0.1, 1, 5, 25, 50 μg/mL). Analysis conditions were same as the in pharmacokinetic evaluation.
3
Quantification of Mycobacterium tuberculosis in Macaque Lungs
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Lung tissues of macaques were harvested and processed for Mtb colony-forming unit determination as described previously [27 (link), 31 (link)]. Briefly, tissue homogenates were made using a homogenizer (PRO 200; PRO Scientific) and were diluted using sterile PBS + 0.05% Tween-80. Fivefold serial dilutions of samples were plated on Middle brook 7H11 plates (Becton Dickinson, Catalog No. 297250). The colony-forming unit counts on plates were measured after 3–4 weeks of culture.
4
RNA Extraction and cDNA Synthesis from Ileum
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The frozen tissue samples of ileum were homogenised in TRIzol (Life Technologies, Paisley, UK) using a Pro Scientific homogeniser model PRO200 (PRO Scientific Inc., Oxford, CT, USA) with 3 s pulses repeated 10 times. RNA was then extracted and cleaned using the RNeasy Plus mini kit (Qiagen Ltd., Manchester, UK).
RNA quality and quantity were determined by measuring the absorbance at 260 nm using a NanoDrop spectrophotometer ND-100 (Thermo Scientific, Waltham, MA, United States), all samples had an absorbance above 1.8. RNA quality was further assessed on an Agilent Bioanalyser 2100 (Agilent Technologies Ireland Ltd., Dublin, Ireland) using the RNA 6000 Nano lab chip kit, all samples had RIN values between 8 and 10. cDNA was synthesised using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. 2 µg of total RNA was reverse transcribed into cDNA using MultiScribe reverse transcriptase. The quantity of converted cDNA was determined by absorbance at 260 nm using a NanoDrop spectrophotometer and stored at – 20 °C.
RNA quality and quantity were determined by measuring the absorbance at 260 nm using a NanoDrop spectrophotometer ND-100 (Thermo Scientific, Waltham, MA, United States), all samples had an absorbance above 1.8. RNA quality was further assessed on an Agilent Bioanalyser 2100 (Agilent Technologies Ireland Ltd., Dublin, Ireland) using the RNA 6000 Nano lab chip kit, all samples had RIN values between 8 and 10. cDNA was synthesised using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. 2 µg of total RNA was reverse transcribed into cDNA using MultiScribe reverse transcriptase. The quantity of converted cDNA was determined by absorbance at 260 nm using a NanoDrop spectrophotometer and stored at – 20 °C.
5
RNA Extraction and qPCR Analysis Protocol
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For experiments utilizing myometrial tissue, frozen samples were mechanically homogenized (PRO200, Proscientific, CT, USA) and total RNA samples were isolated using TRIzol RNA extraction as per manufacturer's protocol (Ambion, TX, USA). For mRNA isolation from cultured cells, Total RNA was isolated from HM6ERMS2 and hTERT-HM cells by using the RNeasy/QIAshredder Mini Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. RNA quality and quantity were determined using Qubit 3 Fluorometer (Life Technologies, MA, USA). 1 µg of total RNA samples were then reverse-transcribed to cDNA using ABI high-capacity cDNA-RT kit (Life Technologies, MA, USA) as per manufacturer's protocol. cDNA was diluted and 10 ng/well was analyzed by qPCR (ABI7500; Life Technologies, MA, USA) using SYBR Green Master Mix as per the manufacturer's instruction (Life Technologies, MA, USA). Human GAPDH was used as an internal control for gene expression normalization. Fold changes in gene expression were calculated using the 2−ΔΔCT method. Primers used in this study were designed using PrimerQuest (IDT) and procured from IDT (Newark, USA). Primer efficiencies were ≥85% with hHM cDNA and specificity was validated using melt curve and electrophoresis analysis of the amplified product. Sequences of all primer sets used in the study are detailed in Supplementary Table S2 (34 (link)).
6
Quantifying Fungal Burden in Mice
7
Isolation and Preparation of Taeniid Cysticerci
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The larval stage parasitic forms of taeniids were used for the present studies. Cysticerci of T. crassiceps ORF strain were recovered after three months of infection, from the peritoneum of experimentally infected BALB/c female mice of 5–7 weeks of age, whereas T. solium cysticerci were dissected from naturally infected pigs. After their recovery, the parasites were washed with PBS (pH 7.2), homogenized (PRO200; Pro Scientific Inc., Oxford, CT, USA) in the presence of ice, and resuspended in cytoskeletal buffer (6.7 mM phosphate, 0.04 M KCl, 1 mM MgCl2, pH 7.4) [21 (link)] complemented with a commercial cocktail of protease inhibitors (Complete Protease Inhibitor Cocktail Tablets; Roche, Mannheim, Germany). Later, parasites were frozen at -70°C until required. The study was approved by the Ethics and Research Committees of the Facultad de Medicina, Universidad Nacional Autónoma de México (#IN216213).
8
Enzymatic Antioxidant Assays in Miscanthus
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For plant material enzymatic assays, a Pro200 homogenizer (Pro Scientific Inc., Oxford, CT, USA) was used to prepare leaf homogenates. Three similar-sized leaves of M. giganteus were taken from each test column after 241–290 days of experiment. For determination of catalase (CAT) activity, 0.06 M sodium phosphate buffer (pH 7.4) was added to homogenates, while 0.05 M carbonate buffer (pH 10.2) was added to determine superoxide dismutase (SOD) activity. CAT activity was determined using a static method described by Góth [33 (link)], while SOD activity was measured using method described by Misra and Fridovich [34 (link)]. The homogenates were centrifuged (20 min, 4000 rpm, 4 °C) and then stored at −45 °C until the antioxidant enzyme activity assays were performed.
Measurement of protein quantity and enzymatic activity in the samples was performed using an Evolution 220 spectrophotometer (Thermo Fisher Scientific, Waltham MA, USA) and Insight 2 software (2014) (Thermo Fisher Scientific, Waltham, MA, USA). Before measuring CAT and SOD enzymatic activity, the protein content in the samples was determined using the Bradford method [35 (link)]. The protein concentration of each prepared extract was determined with reference to a standard curve, using bovine albumin as the standard protein. The detail procedure used for enzymatic analysis is described inAppendix A , Appendix A.1.4 .
Measurement of protein quantity and enzymatic activity in the samples was performed using an Evolution 220 spectrophotometer (Thermo Fisher Scientific, Waltham MA, USA) and Insight 2 software (2014) (Thermo Fisher Scientific, Waltham, MA, USA). Before measuring CAT and SOD enzymatic activity, the protein content in the samples was determined using the Bradford method [35 (link)]. The protein concentration of each prepared extract was determined with reference to a standard curve, using bovine albumin as the standard protein. The detail procedure used for enzymatic analysis is described in
9
Plankton DNA Extraction Protocol
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Two technical replicates from each sample, consisting of 2 ml of plankton, were centrifuged at 3,000 rpm for 1 min and the supernatant removed. The tissue was homogenized with a Bio‐Gen PRO200 tissue homogenizer (PRO Scientific, Oxford, CT, USA) for 30 s at the lowest speed (5,000 rpm). The homogenate was centrifuged at 850 x g for 1 min, and 10–40 mg tissue transferred to a new tube. DNA was extracted using the QIAGEN DNeasy Blood & Tissue kit (QIAGEN, Doncaster, Vic., Australia) by adding 180 μl buffer ATL to the tissue, and following the manufacturer's instructions, incorporating an overnight lysis at 56°C. Extracts were eluted in 2 × 100 μl buffer EB and stored at −20°C. No template controls were extracted and stored in the same manner.
10
Western Blot Protocol for Protein Analysis
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