Adult ticks were identified morphologically on an ice-cold plate using various identification keys; 21 were nymphs, two larvae, and five adult female Hyalomma dromedarii. Adult frozen ticks were divided with a parasagittal section using a sterile scalpel, and one half of each was transferred to buffered saline. A total of 300–500 µL of a virus inactivation solution (DNA/RNA Shield; ZymoResearch, Irvine, CA, USA) was added to each sample, depending on the sample quantity. Feces, organ, and tick samples were homogenized using two 2.8 mm ceramic beads (Bertin Technologies, Montigny-le-Bretonneux, France) and a TissueLyser II (QIAGEN, Hilden, Germany), and then frozen at −80 °C for at least one hour. All the samples were subsequently thawed, vortexed, and centrifuged for 2 min at 6200× g. Automated total nucleic acid extraction was performed using 200 µL of each supernatant and QIAamp 96 Virus QIAcube HT Kit on a QIAcube HT device (plate format), or 150 µL and QIAamp Viral RNA Mini QIAcube Kit on a QIAcube machine (for 12 samples; all from QIAGEN), according to the manufacturer’s instructions.
2.8 mm ceramic beads
Manufactured by Bertin Technologies
Sourced in United States
2.8 mm ceramic beads are small, spherical objects made of ceramic material. They have a diameter of approximately 2.8 millimeters. The core function of these beads is to serve as a general-purpose laboratory equipment item.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (2 mentions)
Minilys, by Bertin Technologies (2 mentions)
Qiaamp viral rna mini qiacube kit, by Qiagen (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Dna rna shield, by Zymo Research (1 mentions)
Qiacube machine, by Qiagen (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
4 protocols using 2.8 mm ceramic beads
1
Tick Identification and Nucleic Acid Extraction
2
Ocular Tissue Homogenization Protocol
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All of the animals were euthanized with an injection of a ketamine and xylazine mixture, and the eye balls were immediately enucleated. After removing the extraocular tissue, each eye ball was transferred into a 2 mL Precellys homogenization tube containing five 2.8-mm ceramic beads (Bertin Technologies, Rockville, Maryland) and immersed in 200 μl of homogenization buffer supplemented with 1× protease inhibitor (Roche, Mannheim, Germany). The samples were homogenized using a tissue homogenizer (Minilys®; Bertin Technologies, Rockville, Maryland) at 5,000 rpm for 30 seconds. The homogenized sample was centrifuged at 5,000 g for 5 min at 4 °C, and the resulting supernatants were incubated at 4 °C for 30 min. The samples were subsequently centrifuged at 12,000 g for 20 min at 4 °C and stored at −80 °C prior to additional experiments.
3
Retinal Protein Extraction for Analysis
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Mice were anesthetized using ketamine and xylazine mixture, enucleated immediately and removed the extraocular tissue. Each eye ball was transferred into a 2 ml Precellys homogenization tube containing five 2.8-mm ceramic beads (Bertin Technologies, Rockville, Maryland, USA) and transferred in 100 μl of homogenization buffer with 1 X protease inhibitor (Roche, Mannheim, Germany) and homogenized using a tissue homogenizer (Minilys®; Bertin Technologies, Rockville, Maryland, USA) at 2300 g for 30 s. The homogenized samples were centrifuged at 18,000 g for 5 min at 4 °C, and the resulting supernatants were incubated at 4 °C for 30 min. The samples were subsequently centrifuged at 12,000 g for 20 min at 4 °C and stored at −80 °C.
4
Antioxidant Capacity Analysis of Irradiated Flies
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Four sexually mature males (6 to 8 days old) from each treatment were collected into a 2-ml microcentrifuge tube, snap-frozen in liquid nitrogen, and stored at -80°C until biochemically assayed.
Samples were homogenized using a FastPrep-24 homogenizer (MP Biomedicals, Santa Ana, CA, USA) with 2.8 mm ceramic beads (Bertin Technologies, France) in 500 µl of phosphate buffered saline (PBS) and centrifuged at 5000 g for 5 min at 4°C. The soluble protein concentration in the supernatant of each pool of four males was measured using a BCA kit (ThermoFisher, Rockford, IL, USA), and the concentration of each sample was adjusted to 2 µg/ml of soluble protein. The difference in total antioxidant capacity of irradiated and non-irradiated WT and SOD2 5.2 males was analyzed using a general linear mixed model (GLMM) with type III sums of squares. Line (WT and SOD2 5.2), radiation (0 Gy and 70 Gy), low-oxygen atmosphere treatments (Nx, Hx, and SHx), and their interactions were modeled as xed effects. Block, representing three temporal cohorts, was included as a random effect in the model. GLMM was performed using the lme4 package. Differences between treatments were determined using least-square means (Student's t-test) from lsmeans package. All data analyses in this study were performed using R.
Samples were homogenized using a FastPrep-24 homogenizer (MP Biomedicals, Santa Ana, CA, USA) with 2.8 mm ceramic beads (Bertin Technologies, France) in 500 µl of phosphate buffered saline (PBS) and centrifuged at 5000 g for 5 min at 4°C. The soluble protein concentration in the supernatant of each pool of four males was measured using a BCA kit (ThermoFisher, Rockford, IL, USA), and the concentration of each sample was adjusted to 2 µg/ml of soluble protein. The difference in total antioxidant capacity of irradiated and non-irradiated WT and SOD2 5.2 males was analyzed using a general linear mixed model (GLMM) with type III sums of squares. Line (WT and SOD2 5.2), radiation (0 Gy and 70 Gy), low-oxygen atmosphere treatments (Nx, Hx, and SHx), and their interactions were modeled as xed effects. Block, representing three temporal cohorts, was included as a random effect in the model. GLMM was performed using the lme4 package. Differences between treatments were determined using least-square means (Student's t-test) from lsmeans package. All data analyses in this study were performed using R.
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