Minilys homogenizer

BALB/c scid mice were purchased from Jackson Laboratories, bred and maintained in specific pathogen-free housing. All mouse procedures were carried out in accordance with protocols approved by the IACUC at Texas A&M University. HCC1419 cells (7×10⁶/mouse) and DU145 cells (5×10⁶/mouse) were implanted subcutaneously in the flanks into 6-8 week old female and male mice, respectively. Cells were suspended in a 1:1 mixture of medium:Matrigel (Corning, Cat. # 354234). For estrogen receptor-positive HCC1419 cells [36 (link)], mice were implanted subcutaneously in the neck with 0.72 mg 60 day release estradiol pellets (Innovative Research of America; Cat. # SE-121) three days prior to cell implantation. Tumor size was determined using calipers every three days. Mice were bled retroorbitally at the indicated times. At the end of each experiment, mice were perfused with PBS containing 10 U/ml heparin and tumors isolated for immunohistochemistry or immunoblotting.
For use in immunoblotting, tumors extracted from mice were lysed in RIPA buffer using a kit from the Peglab (precellys ceramic kit 2.8 mm, Cat. # 91-PCS-CK28) and minilys homogenizer (Bertin Technologies). Resulting lysates were subjected to centrifugation. Supernatants were collected and protein concentrations were determined using the BCA protein assay kit (Pierce, Cat. # 23225).

Brain homogenates from frozen human tissue or TgM83^+/- mice [10% (w/v)] were generated by homogenization in calcium- and magnesium-free phosphate-buffered saline (PBS) using a Minilys homogenizer and CK14 soft tissue homogenizing tubes (Bertin). Homogenates were aliquoted and stored at -80 °C for later analysis. To analyze total protein levels, nine volumes of brain homogenate were combined with one volume of 10X detergent buffer [5% (v/v) Nonidet P-40, 5% (w/v) sodium deoxycholate, prepared in PBS] with added Pierce Universal Nuclease (ThermoFisher #88,701) and Halt Phosphatase Inhibitor (ThermoFisher #784,420) before incubation on ice for 20 min with occasional vortexing. Detergent-extracted brain extracts were clarified via centrifugation at 1,000 × g for 5 min at 4 °C prior to further use.

For DNA extraction, fungal isolates were grown on YPG agar containing 25 µg/mL of tetracycline for 1–2 weeks. Approximately 100 mg of mycelium was transferred to a screw-capped 2 mL microcentrifuge tube containing Lysing Matrix C (MP Biomedicals, CA, USA). Samples were frozen in liquid nitrogen and processed for 30–60 s at intermediate speed in a Minilys homogenizer (Bertin technologies, Montigny-le-Bretonneux, France). DNA was extracted from homogenized samples using the DNeasy Plant kit (Qiagen, Venlo, Netherlands) according to the standard protocol as provided by the manufacturer using both a Qiashredder and a DNA adsorption column. In the last step, genomic DNA from one mycelial sample was eluted from the column in 100 µL of buffer EB (10 mM Tris-Cl, pH = 8.5) and stored at −20 °C. DNA concentration was determined using a NanoDrop One device (Thermo Scientific, Waltham, MA, USA). Prior to use as diagnostic PCR templates, DNA samples were diluted in sterile 1× TE buffer (10 mM TRIS, 1 mM EDTA, pH 8.0).

The posterior half of each brain was equally divided sagittally and one portion (one-fourth of brain) was immediately frozen at liquid nitrogen, and stored at −80 °C for western blot (WB) analyses. Brains were homogenized (Minilys homogenizer, Bertin Technologies) in RIPA lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) and centrifuged at 14 000 rpm for 1 h at 4 °C. For WB analyses, supernatants from cell lysates or homogenized tissue were electrophoretically separated using 10% bicine/tris gel (8 M urea) for proteins less than 5 kD or 10% tris/SDS gels for larger proteins. Electrophoresed proteins were transferred to nitrocellulose membranes (Bio-Rad, Richmond, CA, USA), washed and blocked for 1 h at room temperature in tris-buffered saline containing 5% (w/v) non-fat dry milk (TBS/NFDM). After blocking, membranes were hybridized overnight with various primary antibodies, washed and incubated for 1 h with the appropriate HRP-conjugated secondary antibody in TBS/NFDM. Blots were developed using the luminol reagent (Thermo Fisher Scientific) and densitometry analysis was performed using an ImageJ software (Java 1.6.0_20, NIH, USA) as used previously.^{59 (link), 60 (link)}

To recover bacteria, tissues were removed from the wells and washed with 500 μL of PBS. Tissue sections were then transferred in sterile homogenization tubes (Fisherbrand) with 5 mm glass beads (Merck) and 500 μL of PBS. Samples were homogenized using a Minilys Homogenizer (Bertin) for 20 s at 15 m/s. Tissue homogenates were serially diluted in PBS and plated on LB agar. Colony counts were performed after 24 h at 37°C.

The effects of 0,
0.5, and 20 mg/L fluoxetine on CYP gene expression over time were
analyzed with quantitative real-time PCR (qRT-PCR). 1500 worms were
collected in eppendorf tubes with 300 μL of RLT lysis buffer
(Qiagen, Hilden, Germany) and homogenized using a Minilys homogenizer
(Bertin Technologies) at middle speed four times for 20 s and kept
on ice in between. Total RNA was isolated with the QIAshredder and
RNeasy mini kits (Qiagen) according to the manufacturer’s protocol.
The QuantiTect reverse transcription kit (Qiagen) was used for cDNA
generation. RT-qPCR was performed on a Biorad CFX Opus 384 System
using an iQ SYBR Green Supermix (Biorad) for amplification. The gene
of interest was cyp35-a2, and cdc-42 was used as a housekeeping gene. Primers were generated by Biolegio
(Nijmegen, The Netherlands); further details on the RT-qPCR and primers
can be found in SI A7. Three biological
replicates were used for each treatment.

For DNA isolation cells were cultivated on Medium 1 in the absence of minerals under optimal conditions, collected by centrifugation at 9 kG for 15 min and disrupted with glass beads using Minilys homogenizer (Bertin Technologies). DNA was extracted using QIAamp DNA mini kit (Qiagen).