At various time points after injection of S. typhimurium A1-R, mice were sacrificed by CO2 narcosis at 1 h, 1 day, 3 days, 7 days, 14 days and 28 days post-injection, and their blood (0.1 ml), brain, heart, lungs, liver, spleen, kidneys, muscle, bone, small and large intestines, and tumor were harvested. Tissues were removed, weighed, and homogenized in phosphate buffered saline (PBS), at 2 ml per 200 mg of tissue using an homogenizer (Fisher Scientific, USA) at a speed of ˜24,000 rpm for 30-60 s. The resulting suspension was serial diluted by 10× dilutions in PBS and carefully spread onto LB agar. Blood was obtained from intracardiac puncture and mixed with 9 volumes of PBS, after which a 100 μl suspension was spread on LB agar plates. LB agar plates were incubated for 18 h at 37°C. Following incubation, plates were removed from the incubator and colonies were counted. At least three mice were used for each time point. S. typhimurium A1-R-GFP colonies were visualized with an Olympus OV100 Small Imaging System with a CCD camera [39 (link)].
Homogenizer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Homogenizer is a laboratory equipment used to break down and mix solid or viscous samples into a uniform, homogeneous suspension. It operates by applying mechanical force to the sample, ensuring thorough mixing and disruption of the material.
Automatically generated - may contain errors
Lab products found in correlation
Tri reagent, by Thermo Fisher Scientific (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Reverse transcriptase, by Promega (1 mentions)
Rt buffer, by Promega (1 mentions)
Gotaq polymerase, by Promega (1 mentions)
Protease and phosphatase inhibitor, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Imagequant las4000 luminescent imager, by GE Healthcare (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Branson sonifier 250, by Avantor (1 mentions)
Complete protease inhibitors and phosstop, by Roche (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Bullet blender, by Next Advance (1 mentions)
17 protocols using homogenizer
1
Quantifying S. typhimurium Dissemination
2
Quantifying Gene Expression in BV2 Cells and Brain Tissue
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BV2 cells (7.5×105 cells per well in a 6-well plate) were treated with poly(I:C) in the presence or absence of galangin, and total RNA was extracted using TRI reagent (Thermo Fisher Scientific). Cortical brain tissue was homogenized using a homogenizer (Fisher Scientific, Pittsburgh, PA, USA), and total RNA was extracted using TRI reagent. For RT-PCR, total RNA (1 μg) was reverse-transcribed in a reaction mixture containing 0.1 μg of random primers, 3 mM MgCl2, 0.5 mM dNTP, 1× RT buffer, and 10 U reverse transcriptase (Promega). The synthesized cDNA was used as a template for the PCR reaction using Go Taq polymerase (Promega) and primers for the target gene. The primers used in the PCR reaction are shown in Table 1 .
3
Evaluating Anti-Biofilm Efficacy Against Dual-Bacteria Wound Biofilms
4
Western Blot Analysis of p-ERK1/2 and T-ERK1/2
5
Yeast Growth and Protein Extraction
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Yeast growth
in (Yeast Extract–Peptone–Dextrose)
YPD Broth is meticulously cultivated using a well-defined procedure.
To begin, 50 g of YPD Broth was blended with 1 L of distilled water,
ensuring a precise mixture. This suspension underwent autoclaving
at 121 °C for a duration of 15 min. Following this, yeast cultures
are introduced into detergent-free containers. A brief vortexing was
then carried out to uniformly disperse the yeast cells throughout
the medium. The yeast cultures were subsequently nurtured in a shaking
incubator at 300 rpm.
After yeast cell collection and cleanup
with a PBS, 5 g of yeast cells was suspended in the lysis buffer containing
8 M urea, complete protease inhibitors and PhosSTOP (Roche), and 100
mM ammonium bicarbonate (pH 8.0), followed by incubation on ice for
30 min with periodical vortexing. The cells were lysed for 3 min using
a homogenizer (Fisher Scientific) and then sonicated under a 50% duty
cycle, level 10 output for 20 min on ice with a Branson Sonifier 250
(VWR Scientific). The yeast lysate was centrifuged at 14,000g for 10 min at 4 °C to collect the supernatant containing
extracted proteins. The concentration of total proteins was measured
by a bicinchoninic acid (BCA) kit (Fisher Scientific) according to
the manufacturer’s instructions, and the sample was stored
at −80 °C.
in (Yeast Extract–Peptone–Dextrose)
YPD Broth is meticulously cultivated using a well-defined procedure.
To begin, 50 g of YPD Broth was blended with 1 L of distilled water,
ensuring a precise mixture. This suspension underwent autoclaving
at 121 °C for a duration of 15 min. Following this, yeast cultures
are introduced into detergent-free containers. A brief vortexing was
then carried out to uniformly disperse the yeast cells throughout
the medium. The yeast cultures were subsequently nurtured in a shaking
incubator at 300 rpm.
After yeast cell collection and cleanup
with a PBS, 5 g of yeast cells was suspended in the lysis buffer containing
8 M urea, complete protease inhibitors and PhosSTOP (Roche), and 100
mM ammonium bicarbonate (pH 8.0), followed by incubation on ice for
30 min with periodical vortexing. The cells were lysed for 3 min using
a homogenizer (Fisher Scientific) and then sonicated under a 50% duty
cycle, level 10 output for 20 min on ice with a Branson Sonifier 250
(VWR Scientific). The yeast lysate was centrifuged at 14,000g for 10 min at 4 °C to collect the supernatant containing
extracted proteins. The concentration of total proteins was measured
by a bicinchoninic acid (BCA) kit (Fisher Scientific) according to
the manufacturer’s instructions, and the sample was stored
at −80 °C.
6
Extraction and Analysis of RNA from Diverse Tissues
7
Epididymal RNA Isolation Protocol
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The experimental animals were anesthetized by CO2 stunning at 4 months of postnatal age. The male reproductive tract was exposed through an incision on lower abdomen, and the epididymis was separated from the testis and vas deferens in cold PBS. The epididymis was rapidly dissected into initial segment, caput epididymis, corpus epididymis, and cauda epididymis. Each part of the epididymis washed in fresh cold PBS twice was frozen in liquid nitrogen and stored in –80℃ until utilized for total RNA isolation later.
A process based on phenol-chloroform extraction method was employed to isolate total RNA from the corpus epididymis. Briefly, the tissue was homogenized in total RNA extraction solution (iNtRON Biotech, Sungnam, Korea) with a homogenizer (Fisher Scientific, Pittsburgh, USA). Total RNA was precipitated with isopropanol, and a pellet of total RNA was washed in 70% EtOH and dissolved in DEPC-treated RNase-free dH2O. Concentration and purity of total RNA isolated were evaluated with NanoDrop Lite spectrophotometer (Thermo Scientific, Wilmington, USA), and quality of total RNA was determined by gel electrophoresis. Total RNA samples were directly used for construction of cDNAs.
A process based on phenol-chloroform extraction method was employed to isolate total RNA from the corpus epididymis. Briefly, the tissue was homogenized in total RNA extraction solution (iNtRON Biotech, Sungnam, Korea) with a homogenizer (Fisher Scientific, Pittsburgh, USA). Total RNA was precipitated with isopropanol, and a pellet of total RNA was washed in 70% EtOH and dissolved in DEPC-treated RNase-free dH2O. Concentration and purity of total RNA isolated were evaluated with NanoDrop Lite spectrophotometer (Thermo Scientific, Wilmington, USA), and quality of total RNA was determined by gel electrophoresis. Total RNA samples were directly used for construction of cDNAs.
8
Protein Extraction and Western Blot Analysis
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Total cell lysates were obtained from human or murine cell lines7 . Protein was extracted from flash-frozen tumors using a homogenizer (Fisher Scientific) in lysis buffer (1% Triton X-100, 50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2) on ice. The samples were pulsed for 5 seconds, until the tissue was disintegrated and then incubated on ice for 30 min. In all, 20 μg of lysate was loaded onto a sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel, electrophoresed, and transferred to a nitrocellulose membrane. After blocking with 5% fat-free dry milk (Bio-rad) membranes were probed with primary antibodies listed in Supplementary Table 1 overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies were added to membranes and incubated for 1 h. The Pierce ECL western blotting substrate (Thermo Fisher) was used.
9
Liver Lipid Extraction and Quantification
10
SARS-CoV-2 RNA Extraction and Quantification
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The mouse tissues were mixed with DMEM and homogenized (homogenizer; Thermo Fisher Scientific Inc.) for 5 min. The suspension was centrifuged at 12,000 g and 4 °C for 10 min and the supernatant was conserved. The RNA viruses were extracted with a QIAamp Viral RNA Mini Kit (QIAGEN, Dusseldorf, Germany). The viral RNA was quantified by RT-qPCR with Premix Ex Taq (Takara, Beijing, China) targeting the SARS-CoV-2 N gene. The primer and probe sequences were as follows: NF (5′-GGGGAACTTCTCCTGCTAGAAT-3′); NR (5′-CAGACATTTTGCTCTCAAGCTG-3′); and NP (5′-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3′).
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