Briefly, DNA isolation was performed using the EZ1 Advanced XL methods with the EZ1 DNA Blood Tissue Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's standard protocols, with minor modifications. A volume of 200 µl of blood pellet was pretreated by Fast Prep Lysing Matrix B (MP Biomedicals, Eschwege, Germany) in a 2-ml tube containing 1.4-mm ceramic spheres (MP Biomedicals) and 500 µl of lysis buffer (NucliSENS easyMAG, bioMérieux, Marcy-l’Étoile, France). Extraction was repeated in the event of a negative result after quantitative polymerase chain reaction (qPCR), which indicates the presence of PCR inhibitors in the sample, or samples were diluted to 1/20.
1.4 mm ceramic spheres
Manufactured by MP Biomedicals
Sourced in United States
The 1.4 mm ceramic spheres are inert, solid ceramic beads with a uniform diameter of 1.4 millimeters. They are composed of an aluminum oxide (Al2O3) ceramic material. These spheres are designed for use in various laboratory applications that require a consistent, non-reactive solid particulate.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Fastprep 24 5g instrument, by MP Biomedicals (2 mentions)
Scan speed 32 speed vacuum concentrator, by Labogene (2 mentions)
Nucleospin mirna isolation kit, by Macherey-Nagel (2 mentions)
Lysing matrix d tube, by MP Biomedicals (2 mentions)
Fastprep lysing matrix b, by MP Biomedicals (1 mentions)
Nuclisens easymag, by bioMérieux (1 mentions)
Rnase free pbs, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Superscript 4 vilo master mix, by Thermo Fisher Scientific (1 mentions)
Qubit rna br assay kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Fastprep 24 instrument, by MP Biomedicals (1 mentions)
Fastprep tube, by MP Biomedicals (1 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
7 protocols using 1.4 mm ceramic spheres
1
Blood DNA Extraction Protocol
2
RNA Isolation from Ileum Tissue
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Approximately 20 mg of ileum samples stored in RNAlater Stabilization Solution were rinsed twice with RNase-free PBS (Invitrogen). Tissues samples were put in tubes containing 700 mg of 1.4 mm ceramic spheres (MP Biomedical) and 1 ml of TRIzol Reagent (Invitrogen). A FastPrep-24 5G Instrument (MP Biomedical) was used for the mechanic lysis step, consisting of two runs of 30 s at 4 m/s with incubation for 5 min on ice between each run. Tubes were incubated at room temperature for 5 min. A centrifugation was performed at 12,000 × g for 10 min at 4°C and the supernatant was collected to perform RNA isolation according to the manufacturer’s protocol from TRIzol Reagent. RNA purity was confirmed by Nanodrop 1000 (Fisher). RNA was quantified by DeNovix QFX Fluorometer using a Qubit RNA BR assay kit (ThermoFisher Scientific). One microgram of RNA was used for the reverse transcription to cDNA using SuperScript IV VILO Master Mix (Invitrogen) according to the manufacturer’s instructions.
3
Zebrafish Fluorescence Assay and CFU Enumeration
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At 4 dpt, zebrafish embryos were euthanized by an overdose with tricaine (3 mg/ml) and carefully rinsed in PBS. Between 10 and 15 larvae in a volume of 500 µl were transferred into a lysing matrix tube containing 1.4 mm ceramic spheres (MP Biomedical). Homogenization was performed using a FastPrep FP120 cell disrupter (Therma; settings: speed=4, time=10 s) and homogenates were placed on ice for 5 min.
For the plate reader assay, 100 µl of the homogenate was transferred to a black 96-well plate and fluorescence was measured at an absorption wavelength of 550 nm and emission wavelength of 583 nm using a microplate reader (Synergy H1, BioTek).
For CFU plating, the homogenate was transferred to a microcentrifugation tube and passed ten times through a 27 G needle to disrupt mycobacterial clumps. Then, NaOH was added to a final concentration of 1% and incubated at room temperature for 10 min before serial dilutions in PBS were plated on 7H10/10% OADC agar plates containing 10 µg/ml amphotericin B and 25 µg/ml kanamycin A. Plates were incubated at 31.5°C for up to 14 days before CFUs were enumerated.
For the plate reader assay, 100 µl of the homogenate was transferred to a black 96-well plate and fluorescence was measured at an absorption wavelength of 550 nm and emission wavelength of 583 nm using a microplate reader (Synergy H1, BioTek).
For CFU plating, the homogenate was transferred to a microcentrifugation tube and passed ten times through a 27 G needle to disrupt mycobacterial clumps. Then, NaOH was added to a final concentration of 1% and incubated at room temperature for 10 min before serial dilutions in PBS were plated on 7H10/10% OADC agar plates containing 10 µg/ml amphotericin B and 25 µg/ml kanamycin A. Plates were incubated at 31.5°C for up to 14 days before CFUs were enumerated.
4
Liver Protein Extraction and Quantification
5
Quantitative Lipidomics of Liver Tissue
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Liver tissue samples were first frozen at −80 °C and then lyophilized. Then, 25 mg of the powder was weighed into FastPrep tubes with lysing matrix D, 1.4 mm ceramic spheres (MP Biomedicals, Santa Ana, CA, USA) and 1 mL of methyl tert-butyl ether: methanol (MTBE: MeOH, 3: 1, v/v) was added. These were extracted 4 × 15 s, the tubes were shaken vigorously between each of the homogenization runs to ensure proper distribution of the sample. Then, 450 µL of dH2O per 25 mg of powder was added to induce phase separation. After vortexing for 10 s, the tubes were centrifuged at 14,000 rpm (21,255× g). Five hundred µL of the upper organic phase was transferred to an Eppendorf microtube Fisherbrand™ (Thermo Fisher Scientific, Waltham, MA, USA), evaporated to dryness and stored at −80 °C until analysis. Prior to analysis, the stored samples were resuspended in 500 µL iPrOH: MeOH: H2O (65: 30: 5, v/v/v). After centrifugation, 450 µL of the solution was transferred to a vial for ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometric (U-HPLC-HRMS/MS) analysis. All samples were measured in duplicates (in randomized order). To assure the quality of U-HPLC-HRMS/MS measurements, the quality control (QC) samples were prepared (by transferring of 25 µL of each sample into the 4 mL vial), and run together with all of the samples.
6
RNA Extraction from Artery Samples
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The extraction of RNA was performed with the same method for both the microarray and PCR. The RNA was isolated with the Nucleospin miRNA isolation kit (Machery-Nagel, Düren, Germany), following the manufactures instructions for extraction of total RNA. The artery-samples were first homogenized in in Lysing matrix D tubes containing 1.4 mm ceramic spheres (MP Biomedicals, CA, USA) and lysis buffer (ML buffer) from the NucleoSpin kit on dry ice in a FastPrep-24™ 5G instrument (MP Biomedicals, USA) with 3x20sec cycles.
After RNA extraction, the amount of RNA was quantified using a NanoDrop 2000 UV-Vis spectrophotometer (ThermoFisher Scientific, MA, USA). A ratio of sample absorbance at 260 nm and 280 nm in the range of 1.7 to 2.1 was accepted. The RNA which was to be used for microarray analysis was concentrated with a Scan Speed 32 speed vacuum concentrator (Labogene, Denmark). The concentration and quality of the concentrated RNA was determined with a NanoDrop ND1000 spectrophotometer (ThermoFisher Scientific, MA, USA).
After RNA extraction, the amount of RNA was quantified using a NanoDrop 2000 UV-Vis spectrophotometer (ThermoFisher Scientific, MA, USA). A ratio of sample absorbance at 260 nm and 280 nm in the range of 1.7 to 2.1 was accepted. The RNA which was to be used for microarray analysis was concentrated with a Scan Speed 32 speed vacuum concentrator (Labogene, Denmark). The concentration and quality of the concentrated RNA was determined with a NanoDrop ND1000 spectrophotometer (ThermoFisher Scientific, MA, USA).
7
RNA Extraction for Microarray and PCR Analysis
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The extraction of RNA was performed with the same method for both the microarray and PCR. The RNA was isolated with the Nucleospin miRNA isolation kit (Machery-Nagel, Düren, Germany), following the manufactures instructions for extraction of total RNA. The artery-samples were rst homogenized in in Lysing matrix D tubes containing 1.4 mm ceramic spheres (MP Biomedicals, CA, USA) and lysis buffer (ML buffer) from the NucleoSpin kit on dry ice in a FastPrep-24Ô 5G instrument (MP Biomedicals, USA) with 3x20sec cycles.
After RNA extraction, the amount of RNA was quanti ed using a NanoDrop 2000 UV-Vis spectrophotometer (ThermoFisher Scienti c, MA, USA). A ratio of sample absorbance at 260 nm and 280 nm in the range of 1.7 to 2.1 was accepted. The RNA which was to be used for microarray analysis was concentrated with a Scan Speed 32 speed vacuum concentrator (Labogene, Denmark). The concentration and quality of the concentrated RNA was determined with a NanoDrop ND1000 spectrophotometer (ThermoFisher Scienti c, MA, USA).
After RNA extraction, the amount of RNA was quanti ed using a NanoDrop 2000 UV-Vis spectrophotometer (ThermoFisher Scienti c, MA, USA). A ratio of sample absorbance at 260 nm and 280 nm in the range of 1.7 to 2.1 was accepted. The RNA which was to be used for microarray analysis was concentrated with a Scan Speed 32 speed vacuum concentrator (Labogene, Denmark). The concentration and quality of the concentrated RNA was determined with a NanoDrop ND1000 spectrophotometer (ThermoFisher Scienti c, MA, USA).
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