African green monkey kidney epithelial cells (Vero) were cultured in Dulbecco's modified Eagle's medium (BD Biosciences, Franklin Lake, NJ, USA) with 10% fetal bovine serum and 1% penicillin-streptomycin (MP Biomedicals, Santa Ana, CA, USA) at 37°C in a 5% CO2 environment. Three frozen insects from a Rickettsia-positive pool (South Dakota #2) were surface sterilized with 70% ethanol and 10% bleach and placed into tubes of ceramic beads containing 500 μL of cell medium (lysis matrix D; MP Biomedicals). The insects were then macerated at low speed on a BeadBug homogenizer (Benchmark Scientific, Sayreville, NJ, USA) and the homogenate was inoculated into a T25 flask that was ∼50% confluent. Three days after the initial inoculation, the medium was changed to antibiotic free medium and subsequent media changes occurred every 3–4 days. At multiple points following inoculation, culture supernatants and cells were checked for the presence of Rickettsia by PCR and Giemsa staining.
Lysis matrix d
Manufactured by MP Biomedicals
Sourced in United States
Lysis matrix D is a lab equipment product designed for sample preparation. It is used for the disruption and lysis of a variety of sample types, including cells, tissues, and microorganisms. The product provides a physical means of sample homogenization through the use of a matrix material.
Automatically generated - may contain errors
Lab products found in correlation
Fastprep 24 instrument, by MP Biomedicals (2 mentions)
Dulbecco s modified eagle s medium, by BD (1 mentions)
Beadbug homogenizer, by Benchmark Scientific (1 mentions)
Fetal bovine serum (fbs), by MP Biomedicals (1 mentions)
Penicillin streptomycin, by MP Biomedicals (1 mentions)
Magna pure 96 external lysis buffer, by Roche (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Dc kit, by Bio-Rad (1 mentions)
Freezone lyophilizer, by Labconco (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
5 protocols using lysis matrix d
1
Cultivating Rickettsia-Infected Insect Cells
2
Fly Homogenization and Bacterial Enumeration
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In each experiment described above, a sample of 3 to 5 female flies was homogenized in 100 µl of the culture medium preferred by each bacterial strain and 100 µl of lysis matrix D (MP Biomedicals) for 30 s on a FastPrep-24 instrument (MP Biomedicals). The homogenate was diluted to 1 ml with culture medium and spiral plated on agar medium using the WASP-2 apparatus (Microbiology International). CFU counts were made with a Protocol 3 colony counter (Microbiology International).
3
SEOV Detection in Diverse Samples
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Lung, kidney and liver tissues were collected in RNAlater (Applied Biosystems, Foster City, CA, USA) and stored at −80 °C. Tissue samples (+/− 25 mg per sample) were disrupted in MagNA Pure 96 External Lysis Buffer (Roche) by using Lysis matrix D (MP Biomedicals, Santa Ana, CA, USA) and Fast Prep FP120 Homogenizer (Thermo Savant, Carlsbad, CA, USA). RNA isolation was performed on blood (200 µL), urine (200 µL), saliva and rectal swabs using a QIAamp Viral RNA kit. All samples were tested by a SEOV–specific real-time RT-PCR as previously described [6 (link)]. Equine Arteritis Virus was added in the lysis buffer to all samples prior to nucleic acid extraction to exclude inhibitory factors in the sample itself as quality assurance for each experimental step [19 (link)]. A rat β-actin control was used as an amplification control by conventional RT-PCR, to confirm the extraction of nucleic acid from tissue material. Only animals with samples positive for β-actin were included.
4
Quantifying Fly Metabolites in Spent Food
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At 5 or 6 days posteclosion, flies were removed from the vial, and a small aliquot of spent food (~25 mg) was removed from the top, taking care to exclude eggs and larvae. The food was lyophilized at −80°C (FreeZone lyophilizer; Labconco) and then weighed to the nearest microgram using a Mettler Toledo MX5 microbalance. The sample was then homogenized in 100 µl of TET buffer with 100 µl of lysis matrix D (MP Biomedicals) for 1 min on a FastPrep-24 instrument (MP Biomedicals). Debris was pelleted by centrifugation, and the supernatant removed and frozen immediately. After thawing the sample on ice, the glucose content was determined by the glucose oxidase method, as implemented previously (28 (link)), and the soluble protein content determined with the Bio-Rad DC kit according to the manufacturer’s instructions. Three replicate vials were tested for each of three biological replicates.
5
Quantifying Bacterial Colonization in Insect Larvae
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For CFU counts, the midguts of 10 larvae (axenic or gnotobiotic) were dissected into the five defined regions of pH in sterile PBS, and collected in 1 ml mMRS medium41 (link). The sample was homogenized with 100 μl lysis matrix D (MP Biomedicals) with shaking for 1 min in a FastPrep-24 instrument with the default settings (MP Biomedicals). The homogenate was assayed for bacterial abundance by spiral plating (on a WASP-2 instrument, Microbiology International) on mMRS-agar, and incubation under aerobic conditions for Acetobacter and under a CO2 atmosphere for Lactobacillus42 (link). The number of CFUs was scored with the Protocol 3 colony counter (Microbiology International). Axenic larvae were assayed as the negative control: none of these homogenates yielded CFUs.
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