Organs from mice were removed and immediately frozen at −80°C on dry ice. RNA from circulating SARS-CoV-2 was prepared from lungs as described previously.2 (link) Lung homogenates were prepared by thawing and homogenizing in lysing matrix M (MP Biomedical) with 500 μL of PBS using an MP Biomedical Fastprep 24 Tissue Homogenizer. RNA was extracted from the supernatants of organ homogenates centrifuged during 10 min at 2,000g using the Qiagen Rneasy kit. The RNA samples were then used to determine viral RNA content by E-specific qRT-PCR. To determine viral RNA content by Esg-specific qRT-PCR, total RNA was prepared using lysing matrix D (MP Biomedical) containing 1 mL TRIzol reagent (ThermoFisher Scientific) and homogenization for 30 s at 6.0 m/s twice using MP Biomedical Fastprep 24 Tissue Homogenizer. The quality of RNA samples was assessed by use of a Bioanalyzer 2100 (Agilent Technologies). Viral RNA contents were quantitated using a NanoDrop Spectrophotometer (ThermoFisher Scientific NanoDrop). The RNA Integrity Number was 7.5–10.0. SARS-CoV-2 E or E sub-genomic mRNA were quantitated after reverse transcription and real-time quantitative TaqMan PCR, using SuperScript III Platinum One-Step qRT-PCR System (Invitrogen) and specific primers and probe (Eurofins), as recently described.3 (link)
Fastprep 24 tissue homogenizer
Manufactured by MP Biomedicals
Sourced in United States
The FastPrep-24 tissue homogenizer is a laboratory instrument designed for the efficient disruption and homogenization of a variety of sample types, including tissues, cells, and other solid materials. It utilizes rapid, high-speed agitation to effectively break down samples, preparing them for further processing and analysis.
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Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Magna lyser beads tubes, by Roche (3 mentions)
Fast prep lysing matrix a tubes, by MP Biomedicals (3 mentions)
Syto 9 green fluorescent nucleic stain, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Ultracel 10k, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Merck Group (2 mentions)
Gel docking system, by Bio-Rad (2 mentions)
174 protein spotted human cytokine antibody array c2000, by RayBiotech (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Fortessa flow cytometer, by BD (2 mentions)
Anti mouse iga pe antibody clone ma 6e1, by Thermo Fisher Scientific (2 mentions)
Lysing matrix d, by MP Biomedicals (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by MP Biomedicals (1 mentions)
High pure viral nucleic acid kit, by Roche (1 mentions)
Rotor gene q real time pcr cycler, by Qiagen (1 mentions)
33 protocols using fastprep 24 tissue homogenizer
1
Quantification of SARS-CoV-2 RNA from Mouse Lungs
2
Whole Blood Assay for TB Drugs
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A 70 ml sample of whole blood was drawn from each of 4 healthy volunteers who had no history of chronic disease, no history of TB and who had a negative IGRA.
WBA cultures were set-up as described above, but with a 25-fold increase in volumes (to a total volume of 15 ml blood culture) whilst maintaining the ratios of blood and TB inoculum and the drug concentration unchanged. Cultures for the same eight drug/control conditions as described above were prepared in quadruplicate (each replicate assay prepared separately from the blood of one of the four healthy volunteers) and were incubated for 1 h (time selected from the kill curves, as above).
RNA was extracted from 15 ml whole blood cultures by adding Guanidium Thiocyanate solution for host cell lysis, followed by resuspension of cells in 1 ml TRIzol Reagent (Thermo Fisher Scientific, Massachusetts, USA) for Mtb inactivation, Mtb cells were mechanically lyzed using the FastPrep 24 Tissue Homogenizer (MP Biomedicals, California, USA) at 4x30s, 6.0 m/s followed by phenol-chloroform steps outlined in the TRIzol user manual. Samples were treated with DNAse-I using TURBO DNA-free kit (Thermo Fisher Scientific, Massachusetts, USA).
WBA cultures were set-up as described above, but with a 25-fold increase in volumes (to a total volume of 15 ml blood culture) whilst maintaining the ratios of blood and TB inoculum and the drug concentration unchanged. Cultures for the same eight drug/control conditions as described above were prepared in quadruplicate (each replicate assay prepared separately from the blood of one of the four healthy volunteers) and were incubated for 1 h (time selected from the kill curves, as above).
RNA was extracted from 15 ml whole blood cultures by adding Guanidium Thiocyanate solution for host cell lysis, followed by resuspension of cells in 1 ml TRIzol Reagent (Thermo Fisher Scientific, Massachusetts, USA) for Mtb inactivation, Mtb cells were mechanically lyzed using the FastPrep 24 Tissue Homogenizer (MP Biomedicals, California, USA) at 4x30s, 6.0 m/s followed by phenol-chloroform steps outlined in the TRIzol user manual. Samples were treated with DNAse-I using TURBO DNA-free kit (Thermo Fisher Scientific, Massachusetts, USA).
3
Detection of Seal Parvovirus in Spleen Tissue
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In previous studies of humans infected with parvovirus B19, viral DNA could not be detected in blood samples, but was detected in tissues such as bone marrow, tonsils, synovium, and lymphoid tissues [19] (link), . Since replication of SePV was observed in vitro in seal bone marrow cells, this might be also applicable for SePV [12] (link). However, since bone marrow samples were not available, spleen tissue samples were selected. These samples were homogenized in 1 ml Hank's minimal essential medium (HMEM) containing 0.5% lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 µg/ml streptomycin, 100 U/ml polymyxin B sulfate, 250 µg/ml gentamycin, and 50 U/ml nystatin (ICN Pharmaceuticals) (transport medium) using a Fastprep-24 Tissue Homogenizer (MP Biomedicals) and centrifuged briefly. Total nucleic acids were extracted from serum (50 µl) and homogenized spleen tissue (200 µl) using the High Pure Viral Nucleic Acid kit (Roche) according to the instructions of the manufacturer. Samples were screened for SePV DNA using a real-time PCR targeting the VP1 gene as described previously [12] (link). Each sample was tested at least in two independent experiments and to avoid false positive samples due to cross-contamination during mass necropsies, only samples that yielded a cycle threshold (Ct) value below 35 were considered positive.
4
Quantifying Transcript Expression in Mouse Adipose Tissue
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Total RNA was obtained from frozen mouse white adipose tissue via mechanical homogenization at 4.5 m/sec for 30 sec using a FastPrep‐24 tissue homogenizer (MP Biomedicals, Santa Ana, CA, USA) and ceramic beads, followed by guanidinium thiocyanate–phenol–chloroform extraction. RNA was treated with DNase I (Cat# 18068‐015; Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was prepared using 1000 ng total RNA and SuperScript III Reverse Transcriptase (Cat# 18080‐044; Thermo Fisher Scientific, Waltham, MA, USA). Transcript expression was measured using TaqMan Assays with AmpliTaq Gold DNA polymerase (Cat# N8080247; Thermo Fisher Scientific, Waltham, MA, USA) in a Rotor‐Gene Q real‐time PCR cycler (QIAGEN, Hilden, Germany), and target genes were compared to the geometric mean of Rplp0 and Rn18s housekeeping genes using the ΔΔCT method. Gene expression was analyzed in WT mice treated with vehicle (n = 7), WT mice treated with fucoidan (n = 5), SRA−/− mice treated with vehicle (n = 6) and SRA−/− mice treated with fucoidan (n = 8).
5
Antioxidant Enzyme Activity in Aspergillus niger
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A. niger mycelium grown on PDA media were suspended in 50 mM ice-cold phosphate buffer (pH 7.8, 1 mM EDTA and 1 mM PMSF) and an equal volume acid-washed glass beads (0.4–0.6 mm diameter, Sigma) were added to 1 mL of A. niger suspension in an Eppendorf tube. The cells were broken in a FastPrep-24 Tissue Homogenizer (MP Biomedicals, USA) for 40 s at 5500 rpm, repeated four times with 2 min on ice between intervals. The mixture was centrifuged at 12,000 rpm at 4°C for 10 min to obtain the supernatant. Activities of SOD and CAT were determined by procedures described by Giannopolitis and Ries [28] (link) and Beers and Sizer [29] (link) respectively. One SOD unit was defined as the amount of enzyme that inhibits the rate of nitroblue tetrazolium (NBT) reduction by 50% and one unit of CAT was defined as a decrease of 0.1 OD at 240 nm. Activity was expressed as U·mg−1 protein. The protein content was measured according to Bradford [30] (link) using bovine serum albumin as standard.
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A. niger mycelium grown on PDA media were suspended in 50 mM ice-cold phosphate buffer (pH 7.8, 1 mM EDTA and 1 mM PMSF) and an equal volume acid-washed glass beads (0.4–0.6 mm diameter, Sigma) were added to 1 mL of A. niger suspension in an Eppendorf tube. The cells were broken in a FastPrep-24 Tissue Homogenizer (MP Biomedicals, USA) for 40 s at 5500 rpm, repeated four times with 2 min on ice between intervals. The mixture was centrifuged at 12,000 rpm at 4°C for 10 min to obtain the supernatant. Activities of SOD and CAT were determined by procedures described by Giannopolitis and Ries [28] (link) and Beers and Sizer [29] (link) respectively. One SOD unit was defined as the amount of enzyme that inhibits the rate of nitroblue tetrazolium (NBT) reduction by 50% and one unit of CAT was defined as a decrease of 0.1 OD at 240 nm. Activity was expressed as U·mg−1 protein. The protein content was measured according to Bradford [30] (link) using bovine serum albumin as standard.
6
Tissue Homogenization and Protein Extraction
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Whole brain homogenates were lysed using a T25 ULTRA-TURRAX (IKA) with three 30 s pulses in 5 mL tissue lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM PMSF, Phosphatase Inhibitor Cocktail II (Sigma), Phosphatase Inhibitor Cocktail III (Sigma), Protease Inhibitors (Roche Diagnostics). Crude lysates were centrifuged three consecutive times for 12 min at 3,000 x g and 4°C, the supernatant was transferred to a new tube after each centrifugation step. Dissected cortex, striatum and musculus tibialis anterior samples were homogenized in 250–400 μL tissue lysis buffer using lysing matrix M tubes (MP Biomedicals), striatal samples of ZFP-treated mice were homogenized in 80 μL of tissue lysis buffer using Precellys CK14 lysing tubes (Berlin Technologies) in a FastPrep-24 tissue homogenizer (MP Biomedicals) with three 30 s cycles. Crude lysates were centrifuged three consecutive times for 10 min at 16,000 rcf and 4°C, and the supernatant was transferred to a new tube after each centrifugation step. For all tissue homogenates, the total protein concentration was determined using the bicinchoninic acid assay (BCA; Thermo Scientific) and adjusted to 1.5, 2, or 5 mg/mL with tissue lysis buffer. Homogenates were aliquoted, snap-frozen, and stored at -80°C.
7
Diagnostic Protocol for Australian Bat Lyssavirus
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Clinical specimens were collected from flying foxes, submitted to the Australian Centre for Disease Preparedness (ACDP) for the diagnosis of ABLV during 2013 to 2021 from South Australia, Victoria, New South Wales, Queensland, and Western Australia. Tissues were prepared as 10% (w/v) homogenates in Dulbecco's PBS (ph 7.6; Oxoid) containing antibiotics (Sigma-Aldrich) using 1-mm silicon carbide beads (BioSpec Products) in a FastPrep24 tissue homogenizer (MP Biomedical). Samples were clarified by low speed centrifugation (1000×g, 5 min, 4 °C) and the supernatant used for nucleic acid extraction and virus isolation.
ABLV diagnostic testing was undertaken on brain tissue collected from the flying foxes using the fluorescent antibody test (FAT) and quantitative RT-PCR (qRT-PCR), as previously described [12 (link)].
ABLV diagnostic testing was undertaken on brain tissue collected from the flying foxes using the fluorescent antibody test (FAT) and quantitative RT-PCR (qRT-PCR), as previously described [12 (link)].
8
Protein Expression and Cytokine Profiling
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Tumor-containing tibiae and subcutaneous tumors were snap-frozen in liquid nitrogen. Frozen tibiae were homogenized in RIPA buffer (50 (link)) using a FastPrep-24 tissue homogenizer (MP Biomedicals). Prostate cancer cell lines were lysed using RIPA buffer as described previously (50 (link)). Total protein (10–20 μg) was separated by SDS-PAGE and transferred to PVDF membranes (Fisher). Membranes were blocked with 5% BSA-TBST and incubated with antibodies diluted in 5% BSA-TBST as previously described (50 (link)). Primary antibodies were detected using HRP-conjugated secondary antibodies (Sigma) by chemiluminescence using Quantity One imaging software on a Bio-Rad Gel Docking system. Antibodies listed in Supplementary Table S1. Equal amounts of protein from subcutaneous or tibiae tumors pooled from two mice were incubated with a 174-protein spotted human cytokine antibody array C2000 (Ray Biotech Inc) according to the manufacturer’s protocol.
An MMP-3 ELISA (R&D) that recognizes both the proenzyme and active form was performed on 24 h serum-starved PC3-NICD3 cells treated with or without Dox. Media were collected and concentrated (Ultracel-10k; Millipore) and equal volumes were used to quantify total MMP-3.
An MMP-3 ELISA (R&D) that recognizes both the proenzyme and active form was performed on 24 h serum-starved PC3-NICD3 cells treated with or without Dox. Media were collected and concentrated (Ultracel-10k; Millipore) and equal volumes were used to quantify total MMP-3.
9
Burn Wound Infection Model with P. aeruginosa
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As described above, the skins were cut into discs, betadine treated, washed with PBS, tap dried with sterile gauze, burned, treated with 40 mM CeN, and incubated for 2 days at 37°C. The burned-skin discs were then inoculated with ∼106 CFU of P. aeruginosa-1244 per skin disc and 4 h later were overlaid with Acticoat dressing (Smith & Nephew) that was wetted with sterile H2O. After incubation for 2 days at 37°C, Acticoat was removed and the burned-skin discs were homogenized in 1 mL PBS in MagNA Lyser Beads tubes (Roche) using a FastPrep-24 Tissue Homogenizer (MP Biomedicals, LLC). The homogenized samples were plated on P. aeruginosa isolation agar, incubated at 37°C for 18 h, and the viable CFU of skin discs was determined using an automated colony (as described in above section).
10
Specimen Collection and Processing for Viral Analysis
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Swabs and feces were collected in virus transport media (500 μl) (Munster et al., 2009 (link)). Blood samples were collected in blood collection tubes (Minicollect, Greiner Bio-one, 450533, The Netherlands), centrifuged for 10 min at 250 x g and the serum was stored at -20 °C. All organs collected during necropsy were snap frozen and stored at -80 °C. Organs and tumor tissues were supplemented with Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, The Netherlands) and PSG and subsequently homogenized using a FastPrep 24 tissue homogenizer (MP Biomedicals, The Netherlands). Homogenized samples were centrifuged for 10 min at 2000 x g and supernatant was stored at -80 °C or 200 μl was used for Ribonucleic acid (RNA) isolation.
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