Salmonella DNA was isolated in a QIAcube HT robot using the QIAamp 96 DNA QIAcube HT Kit (Qiagen, Valencia, CA). A single Salmonella colony was suspended into 5 ml of TSB and incubated overnight at 37 °C. From the suspension culture, 1 ml was transferred into a 1.2 ml micro-collection tube and centrifuged at 4,000 rpm for 15 minutes at room temperature. After the supernatant was removed, the pellet was re-suspended in ATL buffer (Qiagen) mixed with reagent DX (Qiagen). One tube of small pathogen lysis beads (Qiagen) was mixed with the suspension, and disrupted with the Qiagen TissueLyser system (Qiagen) at 25 Hz, for 5 minutes. The tubes were briefly centrifuged and 40 μl of Proteinase K was added to each tube. The tubes were incubated at 56 °C for 1 hour at 900 rpm in a ThermoMixer (Eppendorf, Hauppauge, NY) followed by a heat shock for 10 minutes at 95 °C. The suspension was cooled to room temperature and 4 μl of RNAse A was added. The prepared samples were set in the QIAcube HT for DNA extraction using a modified protocol provided by Qiagen. The quality of the DNA was determined by the 260/280 ratios on the FLUOstar Omega Microplate Reader (BMG LABTECH, Cary, NC). The DNA quantity was measured with a Quant-iT™ Pico Green® ds DNA Assay kit (Thermo Fisher Scientific) and the DNA was stored at −20 °C until future use.
Tissuelyser system
Manufactured by Qiagen
Sourced in Germany, United States, France
The TissueLyser system is a high-throughput bead-milling device designed for efficient disruption and homogenization of biological samples, such as tissue, plant, and microbial samples. The system utilizes mechanical shaking to process multiple samples simultaneously, enabling rapid and reproducible sample preparation for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
5 mm stainless steel bead, by Qiagen (4 mentions)
Qiaamp dna stool mini kit, by Qiagen (4 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Dynamo flash sybr green qpcr kit, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Qiazol, by Qiagen (2 mentions)
Ampigene cdna synthesis kit, by Enzo Life Sciences (2 mentions)
Ampigene qpcr green mix hi rox, by Enzo Life Sciences (2 mentions)
Psa ncam elisa kit, by Eurobio Scientific (2 mentions)
Zirconia glass beads, by Carl Roth (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Rneasy lipid tissue mini kit, by Qiagen (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
59 protocols using tissuelyser system
1
Salmonella DNA Isolation Protocol
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Salmonella DNA was isolated in a QIAcube HT robot using the QIAamp 96 DNA QIAcube HT Kit (Qiagen, Valencia, CA). A single Salmonella colony was suspended into 5 ml of TSB and incubated overnight at 37 °C. From the suspension culture, 1 ml was transferred into a 1.2 ml micro-collection tube and centrifuged at 4,000 rpm for 15 minutes at room temperature. After the supernatant was removed, the pellet was re-suspended in ATL buffer (Qiagen) mixed with reagent DX (Qiagen). One tube of small pathogen lysis beads (Qiagen) was mixed with the suspension, and disrupted with the Qiagen TissueLyser system (Qiagen) at 25 Hz, for 5 minutes. The tubes were briefly centrifuged and 40 μl of Proteinase K was added to each tube. The tubes were incubated at 56 °C for 1 hour at 900 rpm in a ThermoMixer (Eppendorf, Hauppauge, NY) followed by a heat shock for 10 minutes at 95 °C. The suspension was cooled to room temperature and 4 μl of RNAse A was added. The prepared samples were set in the QIAcube HT for DNA extraction using a modified protocol provided by Qiagen. The quality of the DNA was determined by the 260/280 ratios on the FLUOstar Omega Microplate Reader (BMG LABTECH, Cary, NC). The DNA quantity was measured with a Quant-iT™ Pico Green® ds DNA Assay kit (Thermo Fisher Scientific) and the DNA was stored at −20 °C until future use.
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Salmonella DNA was isolated in a QIAcube HT robot using the QIAamp 96 DNA QIAcube HT Kit (Qiagen, Valencia, CA). A single Salmonella colony was suspended into 5 ml of TSB and incubated overnight at 37 °C. From the suspension culture, 1 ml was transferred into a 1.2 ml micro-collection tube and centrifuged at 4,000 rpm for 15 minutes at room temperature. After the supernatant was removed, the pellet was re-suspended in ATL buffer (Qiagen) mixed with reagent DX (Qiagen). One tube of small pathogen lysis beads (Qiagen) was mixed with the suspension, and disrupted with the Qiagen TissueLyser system (Qiagen) at 25 Hz, for 5 minutes. The tubes were briefly centrifuged and 40 μl of Proteinase K was added to each tube. The tubes were incubated at 56 °C for 1 hour at 900 rpm in a ThermoMixer (Eppendorf, Hauppauge, NY) followed by a heat shock for 10 minutes at 95 °C. The suspension was cooled to room temperature and 4 μl of RNAse A was added. The prepared samples were set in the QIAcube HT for DNA extraction using a modified protocol provided by Qiagen. The quality of the DNA was determined by the 260/280 ratios on the FLUOstar Omega Microplate Reader (BMG LABTECH, Cary, NC). The DNA quantity was measured with a Quant-iT™ Pico Green® ds DNA Assay kit (Thermo Fisher Scientific) and the DNA was stored at −20 °C until future use.
2
Quantifying 11βHSD2 Protein Expression
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Normal human renal and thyroid tissues were homogenized with the Qiagen Tissue Lyser System (QIAGEN, Courtaboeuf, France) and immediately frozen in liquid nitrogen. Then proteins were extracted using RIPA buffer.
Proteins were separated with SDS/PAGE under reducing conditions in the presence of the S-S reducing agent dithiothreitol, then electroblotted onto nitrocellulose membranes and saturated in dry 5% non-fat milk. Membranes were incubated overnight with the primary antibodies (anti-11βHSD2 ABclonal Cat# A8077, RRID:AB_2769875, diluted 1:500, and anti-β-actin ABclonal Cat# AC004, RRID:AB_2737399, diluted 1:50,000).
Primary antibodies were detected with two secondary fluorescent antibodies, an anti-mouse (1:5000, IRDye 680RD) and an anti-rabbit (1:10,000, IRDye 800CW), both from Li-Cor Biosciences, Milan, Italy. Membranes were scanned using the LI-COR Odyssey CLx Imaging System, and the band intensity was quantified with the LI-COR Empiria Studio® Software (v.2.1). Signals were normalised on the ACTB signal and presented as expression relative to a normal human renal specimen (sample 1).
Proteins were separated with SDS/PAGE under reducing conditions in the presence of the S-S reducing agent dithiothreitol, then electroblotted onto nitrocellulose membranes and saturated in dry 5% non-fat milk. Membranes were incubated overnight with the primary antibodies (anti-11βHSD2 ABclonal Cat# A8077, RRID:AB_2769875, diluted 1:500, and anti-β-actin ABclonal Cat# AC004, RRID:AB_2737399, diluted 1:50,000).
Primary antibodies were detected with two secondary fluorescent antibodies, an anti-mouse (1:5000, IRDye 680RD) and an anti-rabbit (1:10,000, IRDye 800CW), both from Li-Cor Biosciences, Milan, Italy. Membranes were scanned using the LI-COR Odyssey CLx Imaging System, and the band intensity was quantified with the LI-COR Empiria Studio® Software (v.2.1). Signals were normalised on the ACTB signal and presented as expression relative to a normal human renal specimen (sample 1).
3
RNA Extraction from Cultured and Patient Cells
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The cultured cells and patient cells were disrupted using a TissueLyser system (Qiagen, Valencia, CA, USA) for 60 s at 25 Hz. Total RNA was extracted using 1 mL of TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. RNA was resuspended in 45 μL of DPEC water and quantified using a NanoDrop One UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Then, RNA was quantified with the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) using a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) according to the protocol. RNA samples were stored at −70 °C until further use.
Then, RNA was quantified with the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) using a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) according to the protocol. RNA samples were stored at −70 °C until further use.
4
Quantitative mRNA Analysis of Mouse Brain
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Brain hemispheres of C57BL/6 mouse, tMCAO mice at 24 h and 48 h post reperfusion were used for total mRNA isolation according to the protocols of the Isol-RNA Lysis Reagent (5 PRIME, Gaithersburg, USA) and the bead-milling TissueLyser system (Qiagen, Hilden, Germany). QuantiTect® Reverse Transcription Kit (Qiagen, Hilden, Germany) was used to generate cDNA. The primers (Microsynth, Balgach, Switzerland) used for the quantitative polymerase chain reaction (qPCR) are summarized in Supplementary Table 1. Quantitation of CNR2, Iba1, TNF-α, MMP9, GFAP and MAP-2 mRNA expression was performed with the DyNAmo™ Flash SYBR® Green qPCR Kit (Thermo Scientific, Runcorn, UK) using a 7900 HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, USA). The amplification signals were detected in real-time, which permitted accurate quantification of the amounts of the initial RNA template during 40 cycles according to the manufacturer's protocol. All reactions were performed in duplicates and in two independent runs. Quantitative analysis was performed using the SDS Software (v2.4) and a previously described 2-ΔΔCt quantification method [69] . The specificity of the PCR products of each run was determined and verified with the SDS dissociation curve analysis feature.
5
Viral Plaque Assay for Detection
6
Quantifying ACE2 and TMPRSS2 Expression in Primate Adipose Tissue
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Fragments of 100–200 μg of frozen SCAT and VAT samples from seven cynomolgus macaques were dissociated in Qiazol (Qiagen, Hilden, Germany) using stainless steel beads in the tissue-lyser system (Qiagen). The RNA-containing aqueous phase was collected after treatment with chloroform. Total RNA from the aqueous phase was precipitated with ethanol. RNA isolation and reverse transcription were performed according to the manufacturers’ instructions (NucleoSpin™ RNA Plus Kit (Macherey-Nagel)) and the enhanced RT-PCR Kit (Sigma-Aldrich), respectively. ACE2 mRNA expression was determined using a standard reverse-transcription PCR assay and commercially available TaqMan probes (Applied Biosystems, Foster City, CA, United States) on the CFX96 thermocycler (Bio-Rad, Hercules, CA, United States). All results were normalized against expression of the PPIA gene. The 18 S rRNA gene was used as second housekeeping gene. The TaqMan probes for ACE2 (Mf01085327_m1), TMPRSS2 (Mf02802837_m1), adiponectin (ADIPOQ Mf02788052_m1), leptin (LEP Mf02788316_m1), PPIA (Mf04932064_gH), and 18 S (Hs99999901_s1) were used.
7
Quantification of Gene Expression in Epididymal Adipose Tissue
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Epididymal adipose tissues were homogenized using the TissueLyser system (#85300, Qiagen, Venlo, The Netherlands), and total RNA was isolated using TRIzol reagent (#15596018, Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA content and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). One microgram of total RNA was reverse-transcribed into cDNA using the AMPIGENE® cDNA synthesis kit (#END-KIT106, Enzo Life Sciences, Farmingdale, NY, USA) and a SimpliAmp Thermal Cycler (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Real-time PCR was performed using AMPIGENE® qPCR Green Mix Hi-ROX (#ENZ-NUC104, Enzo Life Sciences) on a StepOne Plus real-time PCR system (Applied Biosystems). The thermal cycles were as follows: 94 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s. Mouse 18s rRNA was used as a reference gene, and relative gene expression levels were analyzed using the 2−ΔΔCt method. The primers used for the real-time PCR analysis are listed in Supplementary Materials Table S1 .
8
Quantitative Bioanalysis of Compounds
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Two hundred microliters of precipitation solution (i.e., acetonitrile added with 100 ng/mL internal standard, IS) were added to 50 μL of serum, or of muscle or spinal cord lysate [obtained by homogenizing 15 mg of tissue with a tissue lyser system (Qiagen, Hilden, Germany)]. Then, samples were centrifuged at 14,000 rpm for 10 min and the clear supernatant was injected onto the LC‐MS/MS system for analysis. The calibration curve was constructed by plotting the peak area ratio of compound/IS against the nominal concentration of compound standards in control mouse serum or tissue lysates. Following the evaluation of different weighting factors, the results were fitted to linear regression analysis with the use of a 1/x2 (x = concentration) weighting factor. The calibration curves had a correlation coefficient (r) of 0.99 or better.
9
Pathogenic Xanthomonas Screening in Rice
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Rice variety IR24 with its isogenic line IRBB4 were screened using African Xoo strain BAI3 and Asian Xoo strain PXO61. Two, three and four pieces of 5 cm from the apex to the base of infected leaf were harvested 4, 8 and 12 days after inoculation, respectively. On each day, infected leaves fragments were harvested on three different plants. Infected leaves collected were briefly rinsed in 70 % of ethanol for 10 s followed by submersion in sterilized water. Leaves were put into 2 ml eppendorf tubes containing 2 metallic beads (ϕ = 3 mm), frozen by submersion into liquid nitrogen and ground into fine powder using the Qiagen Tissue Lyser system (30 rounds/s for 2 min). Ground material was resuspended in 1 ml of sterilized water and 10 μl drops of a dilution series were spotted onto PSA medium plate in triplicates. The plates were incubated at 28 °C until colonies could be counted. This experiment was performed three times.
10
Quantifying Neutrophil Infiltration via MPO
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A modified version of the study by Binker et al. [25 (link)] was used to assess the activity of MPO as a marker of neutrophil infiltration (studies 3–5). The tissue was weighed and mixed with 0.5 mL 50 mM K- phosphate buffer pH 6.0 with 50 mM hexadecyltrimethylammonium bromide (Sigma-Aldrich, Denmark) in 2 mL Eppendorf tubes containing a 5-mm stainless steel bead (Qiagen, Germany). Samples were homogenised for 6 min at 25 Hz (Qiagen TissueLyser System), and snap frozen on dry ice followed by thawing in water. This step was repeated three times. Samples were centrifuged (16,000 g, 30 min, at room temperature). Supernatants were diluted in 50 mM K-phosphate buffer (pH 6.0) and 10-μL samples were loaded onto a 98-well plate with 190 μL substrate buffer (50 mM K-phosphate buffer, 0.167 mg/mL O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide (H2O2)). The change in absorbance was measured at 450 nm every minute. Changes were checked for linearity and units of MPO/mg were calculated using the extinction coefficient for H2O2 (1.13 x 104 M-1 cm-1) defining one unit as the amount of enzyme that degrades 1 μmol H2O2/min.
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