To determine the infectious ZIKV titer, 6,000 Vero cells were seeded in 100 μL in a 96-well flat-bottom plate and incubated overnight. ZIKV samples from organs of immunized animals were collected on days of killing and frozen at –80°C, until homogenization with lysing matrix M (MP Biomedical, France) in DMEM media containing 2% fetal bovine serum. Approximately 50 mg of frozen tissue samples was homogenized twice using the FastPrep-24 (VWR, France) each cycle for 30 s at 4.0 m/s. The supernatant of homogenized tissue samples was collected after centrifugation to remove debris. The supernatants were titrated 10-fold, and 100 μL of each sample was used for incubation with Vero cells. The titration of ZIKV samples was performed in triplicates, and TCID50/mg was calculated according to Reed and Muench.56
Lysing matrix m
Manufactured by MP Biomedicals
Sourced in Switzerland
The Lysing Matrix M is a specialized laboratory equipment designed for efficient sample homogenization and cell lysis. It is composed of a matrix of ceramic beads and provides a mechanical method for disrupting cells and biological samples to release their contents, such as proteins, nucleic acids, and other intracellular components. The Lysing Matrix M is suitable for use with a wide range of sample types, including microbial cultures, tissues, and plant materials. Its core function is to facilitate the extraction and isolation of target analytes from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin 13e5 rabbit mab, by Cell Signaling Technology (2 mentions)
Tris glycine gel, by Thermo Fisher Scientific (2 mentions)
Fastprep 24 sample disruption instrument, by MP Biomedicals (2 mentions)
Lrp5 d80f2 rabbit mab, by Cell Signaling Technology (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Fastprep24, by Avantor (1 mentions)
Superscript 3 platinum one step qrt pcr system, by Thermo Fisher Scientific (1 mentions)
Lysing matrix d, by MP Biomedicals (1 mentions)
Fastprep 24 tissue homogenizer, by MP Biomedicals (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
4 protocols using lysing matrix m
1
ZIKV Titration in Vero Cells
2
Quantification of SARS-CoV-2 RNA from Mouse Lungs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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)
3
Protein Expression Analysis of LRP5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lack of expression of functional LRP5 protein was confirmed by immunnoblotting.
Protein from rat tail biopsies was isolated in lysis buffer (50 mM Na 2 HPO 4 , 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.5 mM DTT, protease inhibitor tablet Roche, Basal, Switzerland) using lysing matrix M (MP Biomedicals, Irvine, CA) for homogenization in the FastPrep-24 sample disruption instrument (MP Biomedicals). The samples were centrifuged at 21,000 x g for 30 min at 4 °C to pellet nonlysed tissue. We analyzed 40 μg of protein by SDS-polyacrylamide gel electrophoresis on an 8% Trisglycine gel (Thermo Fisher Scientific) and transferred to PVDF western blotting membrane overnight at 4 °C. Immunoblotting was performed using the following antibodies: Lrp5 (D80F2) rabbit mAb (Cell Signaling, 5731), β-actin (13E5) rabbit mAb (Cell Signaling, 5125) (Cell Signalling Technology, Danvers, MA), and V5 Tag mAb, HRPP (Thermo Fisher Scientific, R96125).
Protein from rat tail biopsies was isolated in lysis buffer (50 mM Na 2 HPO 4 , 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.5 mM DTT, protease inhibitor tablet Roche, Basal, Switzerland) using lysing matrix M (MP Biomedicals, Irvine, CA) for homogenization in the FastPrep-24 sample disruption instrument (MP Biomedicals). The samples were centrifuged at 21,000 x g for 30 min at 4 °C to pellet nonlysed tissue. We analyzed 40 μg of protein by SDS-polyacrylamide gel electrophoresis on an 8% Trisglycine gel (Thermo Fisher Scientific) and transferred to PVDF western blotting membrane overnight at 4 °C. Immunoblotting was performed using the following antibodies: Lrp5 (D80F2) rabbit mAb (Cell Signaling, 5731), β-actin (13E5) rabbit mAb (Cell Signaling, 5125) (Cell Signalling Technology, Danvers, MA), and V5 Tag mAb, HRPP (Thermo Fisher Scientific, R96125).
4
Protein Expression Analysis of LRP5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lack of expression of functional LRP5 protein was confirmed by immunnoblotting.
Protein from rat tail biopsies was isolated in lysis buffer (50 mM Na 2 HPO 4 , 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.5 mM DTT, protease inhibitor tablet Roche, Basal, Switzerland) using lysing matrix M (MP Biomedicals, Irvine, CA) for homogenization in the FastPrep-24 sample disruption instrument (MP Biomedicals). The samples were centrifuged at 21,000 x g for 30 min at 4 °C to pellet nonlysed tissue. We analyzed 40 μg of protein by SDS-polyacrylamide gel electrophoresis on an 8% Trisglycine gel (Thermo Fisher Scientific) and transferred to PVDF western blotting membrane overnight at 4 °C. Immunoblotting was performed using the following antibodies: Lrp5 (D80F2) rabbit mAb (Cell Signaling, 5731), β-actin (13E5) rabbit mAb (Cell Signaling, 5125) (Cell Signalling Technology, Danvers, MA), and V5 Tag mAb, HRPP (Thermo Fisher Scientific, R96125).
Protein from rat tail biopsies was isolated in lysis buffer (50 mM Na 2 HPO 4 , 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.5 mM DTT, protease inhibitor tablet Roche, Basal, Switzerland) using lysing matrix M (MP Biomedicals, Irvine, CA) for homogenization in the FastPrep-24 sample disruption instrument (MP Biomedicals). The samples were centrifuged at 21,000 x g for 30 min at 4 °C to pellet nonlysed tissue. We analyzed 40 μg of protein by SDS-polyacrylamide gel electrophoresis on an 8% Trisglycine gel (Thermo Fisher Scientific) and transferred to PVDF western blotting membrane overnight at 4 °C. Immunoblotting was performed using the following antibodies: Lrp5 (D80F2) rabbit mAb (Cell Signaling, 5731), β-actin (13E5) rabbit mAb (Cell Signaling, 5125) (Cell Signalling Technology, Danvers, MA), and V5 Tag mAb, HRPP (Thermo Fisher Scientific, R96125).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!