Whole-cell lysates were prepared using lysis buffer (1% NP-40, 50 mM Tris-HCl pH 8.0, 100 mM sodium fluoride, 30 mM sodium pyrophosphate, 2 mM sodium molybdate, 5 mM EDTA, and 2 mM sodium orthovanadate) containing protease inhibitors (10 μg ml−1 aprotinin, 10 μg ml−1 leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Frozen tumour samples were diced into small pieces in cold lysis buffer on dry ice and homogenised using a Tissue Tearor (Model 398, Biospec Products, Inc., Bartlesville, OK, USA) for 2 s, —three to five times, on ice, and the cell lysate was then rocked overnight at 4 °C. The lysates were cleared by centrifugation at 14 000 g for 20 min at 4 °C, and lysate protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad Laboratories Hercules, CA, USA). Nuclear, membrane, and cytoplasmic fraction lysates were prepared by using a Qproteome Cell Compartment Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. Electrophoresis and western blotting were performed as described previously (Rubin et al, 2001 (link)). The hybridisation signals were detected by chemiluminescence (ECL, Amersham Pharmacia Biotechnology, Marlborough, MA, USA) and captured using a FUJI LAS1000-plus chemiluminescence imaging system.
Tissue tearor model 398
Manufactured by Biospec
Sourced in United States
The Biospec Tissue Tearor (Model 398) is a laboratory instrument designed for the rapid and efficient disruption of biological samples, such as tissue, cells, and other specimens. It operates using a high-speed, bidirectional rotor that rapidly homogenizes the sample, making it suitable for various applications that require the extraction of cellular components or preparation of samples for further analysis.
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2 protocols using tissue tearor model 398
1
Whole-cell Lysate Preparation and Western Blotting
2
Murine Disseminated Candidiasis Model
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A disseminated murine candidiasis model [22] (link) was selected for the assay. Strains SC5314, spt20Δ/Δ, spt20Δ/SPT20 were used to infect mice. Cultures were grown overnight then washed 3 times with PBS. CD-1, six-week old, female mice were infected with 1.5 × 106 CFUs suspended in PBS via a tail vein injection in a 100 µl volume. Twelve mice were inoculated per strain tested and observed daily. Animal survival was evaluated by using the Kaplan-Meier method and differences were determined by using the log-rank test (STATA 6; STATA, College Station, TX). A p value of < 0.05 was considered statistically significant.
Fungal burden in the kidneys was assessed using four mice per infection strain [23] (link). We harvested the kidneys from mice aseptically 2 days post infection. Tissues were weighed and homogenized in sterile PBS by use of a Tissue Tearor (model 398; Biospec Products Inc., Racine, WI). Serial dilutions were plated on YPD agar plates containing 100 µg/mL ampicillin, 100 µg/mL streptomycin, and 45 µg/mL kanamycin. The cfu/g kidney were counted after growth at 30°C for 48 h. Statistical analyses were performed using ANOVA and post hoc (Bonferroni and Student–Newman–Keuls) tests.
Fungal burden in the kidneys was assessed using four mice per infection strain [23] (link). We harvested the kidneys from mice aseptically 2 days post infection. Tissues were weighed and homogenized in sterile PBS by use of a Tissue Tearor (model 398; Biospec Products Inc., Racine, WI). Serial dilutions were plated on YPD agar plates containing 100 µg/mL ampicillin, 100 µg/mL streptomycin, and 45 µg/mL kanamycin. The cfu/g kidney were counted after growth at 30°C for 48 h. Statistical analyses were performed using ANOVA and post hoc (Bonferroni and Student–Newman–Keuls) tests.
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