On day 24 after infection with RSV, mice were euthanized and lungs were collected in 2 ml Dulbecco’s phosphate buffered saline (DPBS) on ice. Lungs were homogenized using a PowerGen 125 Homogenizer (Fisher Scientific) and kept on ice. Serial dilutions of homogenates were made in EMEM with 10% fetal bovine serum (FBS). Virus titrations were in 12 well tissue culture plates that were 80–90% confluent with HEp-2 cells. Briefly, medium was removed from plates and serial dilutions of homogenates were plated in duplicate, 100 μl per well. Plates were incubated at 37°C, 5% CO2 for 1 hour with shaking every 15 minutes. One ml of pre-warmed 0.75% methyl cellulose in EMEM plus 10% FBS was added to each well for further incubation for 5 days. The medium with methyl cellulose was removed by aspiration. Cells were fixed with 1 ml 10% buffered formalin (Fisher Scientific) for 1 hour at room temperature. Formalin was removed and plates were washed with tap water. Then, 1 ml Hematoxylin solution (Sigma-Aldrich) was added for 20 minutes at room temperature. Plates were washed with tap water, air dried, and plaques were counted to determine plaque forming units (pfu) per lung.
Powergen 125 homogenizer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PowerGen 125 homogenizer is a laboratory equipment designed for the homogenization of biological samples. It is a compact and versatile tool that can be used to prepare samples for various analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (4 mentions)
Rt2 first strand kit, by Qiagen (3 mentions)
Iq5 system, by Bio-Rad (3 mentions)
Rt2 master mix kit, by Qiagen (3 mentions)
Picopure rna isolation columns, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Buffered formalin, by Thermo Fisher Scientific (1 mentions)
Hematoxylin solution, by Merck Group (1 mentions)
Sonic dismembrator model 500, by Thermo Fisher Scientific (1 mentions)
Ascent software, by Thermo Fisher Scientific (1 mentions)
Fluoroskan ascent fl microplate fluorometer, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Signosis (1 mentions)
Ripa buffer, by Bio-Rad (1 mentions)
Trizol solution, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Synergy h1 hybrid multi mode reader, by Agilent Technologies (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Viia7, by Thermo Fisher Scientific (1 mentions)
20 protocols using powergen 125 homogenizer
1
Quantification of RSV Viral Titers in Murine Lungs
2
Enumerating Listeria monocytogenes in mice
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L. monocytogenes was administered by oral gavage in all in vivo experiments.
To enumerate L. monocytogenes growth in mouse tissue, collected organs were resuspended in PBS Triton X-100 (Fisher) 0.05%, homogenized for 30″ to 1′ with a Power Gen 125 homogenizer (Fisher Scientific) (power level: 5). Metal probes were washed in between samples through 2 immersions in ethanol and one in PBS for 10–15″ each. Serial dilutions of the homogenates were prepared in PBS Triton and plated on BHI plates supplemented with streptomycin (100 μg/ml) and nalidixic acid (50 μg/ml). Colonies were enumerated after 24–36 h of incubation at 37 °C.
For CFU enumeration in intestinal wall, after excision small and large intestine were separated, cleared of content by squeezing with forceps, cut longitudinally and washed vigorously 4–6 times (10 s vortex or manual shaking) in ice-cold PBS. The washed tissues were then processed as described above.
For CFU enumeration in intestinal content and fecal pellets, starting material was weighted (unless total CFU amount was calculated) and resuspended in PBS to a concentration of 100 mg/ml. Serial dilutions of the original suspension were plated.
To enumerate L. monocytogenes growth in mouse tissue, collected organs were resuspended in PBS Triton X-100 (Fisher) 0.05%, homogenized for 30″ to 1′ with a Power Gen 125 homogenizer (Fisher Scientific) (power level: 5). Metal probes were washed in between samples through 2 immersions in ethanol and one in PBS for 10–15″ each. Serial dilutions of the homogenates were prepared in PBS Triton and plated on BHI plates supplemented with streptomycin (100 μg/ml) and nalidixic acid (50 μg/ml). Colonies were enumerated after 24–36 h of incubation at 37 °C.
For CFU enumeration in intestinal wall, after excision small and large intestine were separated, cleared of content by squeezing with forceps, cut longitudinally and washed vigorously 4–6 times (10 s vortex or manual shaking) in ice-cold PBS. The washed tissues were then processed as described above.
For CFU enumeration in intestinal content and fecal pellets, starting material was weighted (unless total CFU amount was calculated) and resuspended in PBS to a concentration of 100 mg/ml. Serial dilutions of the original suspension were plated.
3
Cytokine Profiling of Tumor Samples
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To prepare the cell lysate, 100 mg of fresh tumor from WT and COL tumor mice was placed in 1 ml of cell lysis buffer (Signosis, Inc., Sunnydale, CA, USA). Tumors were homogenized on ice in a PowerGen 125 homogenizer (Fisherbrand, Pittsburgh, PA, USA) for one minute. Lysates were sonicated at 20 % power for 10 seconds three times using a Sonic Dismembrator Model 500 (Fisher Scientific) with a Branson tip Model 102 converter. The samples were centrifuged at 10,000 RPM for 5 minutes. The supernatant was then aliquoted and frozen at −80 °C until ready to use.
Mouse Cytokine ELISA Plate Array (Chemiluminescence) (Signosis, Inc.) is a 96-well plate divided into four sections; each section has three columns for one sample. Each section also has 23 specific cytokine capture antibodies coated onto each well and one blank well. Samples were thawed and diluted to 10 μg per 100 μl per well. Briefly, each well was incubated with 100 μl of sample for 2 hours, followed by biotin-labeled antibody mixture for 1 hour with gentle shaking, then with strepavidin-HRP conjugate for 30 minutes with gentle shaking, then finally with substrate solution for 2 minutes. The plate was read on a Fluoroskan Ascent™ FL microplate fluorometer using the Ascent™ Software (Thermo Scientific, Waltham, MA, USA).
Mouse Cytokine ELISA Plate Array (Chemiluminescence) (Signosis, Inc.) is a 96-well plate divided into four sections; each section has three columns for one sample. Each section also has 23 specific cytokine capture antibodies coated onto each well and one blank well. Samples were thawed and diluted to 10 μg per 100 μl per well. Briefly, each well was incubated with 100 μl of sample for 2 hours, followed by biotin-labeled antibody mixture for 1 hour with gentle shaking, then with strepavidin-HRP conjugate for 30 minutes with gentle shaking, then finally with substrate solution for 2 minutes. The plate was read on a Fluoroskan Ascent™ FL microplate fluorometer using the Ascent™ Software (Thermo Scientific, Waltham, MA, USA).
4
Placenta Tissue Sampling and Preservation
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Term placentae were sampled within an hour of delivery to preserve specimen integrity. Large tissue biopsies (approximately 2.5 cm3) were dissected from central and peripheral cotyledons. Tissue samples were further dissected into small pieces (~1 cm3), then placed in cryovials and flash‐frozen in liquid nitrogen. Frozen placenta tissue was powdered on ice and homogenized in radioimmunoprecipitation assay (RIPA) buffer (BioRad) using Powergen 125 homogenizer (Fisherbrand). The protein lysate was centrifuged at 1000 g for 10 min at 4°C and stored at −80°C until further analysis. Full‐thickness histological samples were taken from healthy cotyledons of the placenta, avoiding areas of necrosis and calcification. Sections were rinsed in non‐sterile phosphate‐buffered saline (PBS). For formalin‐fixed, paraffin‐embedded samples (FFPE), the dissected sections were placed in 10% formalin for 48 h, then rinsed three times in non‐sterile PBS and subsequently preserved in paraffin blocks. Additional sections were placed into plastic cryomolds containing O.C.T embedding compound. Cryomolds were wrapped in aluminum foil and flash‐frozen in liquid nitrogen.
5
Placental Tissue Sampling and Protein Isolation
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Within an hour of vaginal delivery or caesarian section, term placentae were collected and the umbilical cord and chorioamniotic membranes were removed. All sampling was performed on ice and avoided areas of excess calcifications, abruptions, or necrosis. Tissue samples of approximately 2.5 cm3 were taken from both central and peripheral cotyledons and then divided into smaller pieces of approximately 1 cm3. Dissected samples were placed into cryovials and flash-frozen in liquid nitrogen for transport, then stored at −80 °C. To isolate placenta protein lysate, frozen samples were powdered on ice and homogenized with the Powergen 125 homogenizer (Fisherbrand, Pittsburg, PA, USA) in radioimmunoprecipitation buffer (BioRad, Hercules, CA, USA). Lysate underwent 1000× g centrifugation at 4 °C for 10 min to remove debris. In addition to tissue samples, full-thickness histological samples were taken from the fetal side of healthy cotyledons. Sections were fixed in formalin for 48 h before being embedded in paraffin blocks.
6
Quantitative RT-PCR Analysis of Inflammatory Markers in Mouse Liver
7
Ileal Biopsy RNA Extraction
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Fresh frozen ileal biopsies were homogenized individually in 2 ml of Trizol solution (Life Technologies) with the PowerGen125 homogenizer (Fisher Scientific) and 1 ml aliquots placed into 1.5 mL microcentrifuge tubes. RNA was subsequently extracted using phenol/chloroform extraction methods as previously described[58 (link)]. The RNA was reconstituted in 50ul of RNA Storing Solution (Life Technologies) and stored at -800 C until batch analysis.
8
Comprehensive Total RNA Extraction
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Total RNA was extracted using TRIzol Reagent (Life Technologies, cat. #15596026) and RNeasy Mini Kit (Qiagen, cat. #74104) or RNeasy Micro Kit (Qiagen, cat. #74004). Briefly, 10–25 mg frozen tissue was placed into 50‐ml conical tubes and 1 ml TRIzol added. The mixture was placed in a PowerGen 125 homogenizer (Fisher Scientific) for 20–40 s, or a Kontes Pestle Cordless Motor (Fisher Scientific) for smaller samples. Homogenate was left for 5 min at room temperature and then 200 µl chloroform added to the tube and vortexed for 15 s. After Leaving the homogenate for 2 min at room temperature, the sample was transferred into a Phase Lock Gel Heavy tube (5 Prime, yellow‐color tube, spun 20 s prior to use). Samples were centrifuged at 12,000 g for 10 min at 4°C to separate phases, and subsequently, the upper aqueous phase was transferred to a new tube. One volume of 70% ethanol was added and mixed by pipetting. Samples were subsequently pipetted into an RNeasy mini‐column and the RNeasy Mini Kit procedure for RNA isolation was performed per manufacturer recommendations.
9
Mouse Liver Gene Expression Analysis
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Mouse liver tissues were mechanically homogenized using the PowerGen 125 Homogenizer (Fisher Scientific, Fair Lawn, NJ). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA). Total RNA (500 ng) was converted into cDNA using the RT2 First Strand Kit (SA Biosciences, Valencia, California). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the RT2 Master Mix Kit (SA Biosciences, Valencia, CA) and the iQ 5 system (Bio-Rad Laboratories, Hercules, CA). Quantitative RT-PCR was performed for mRNA expression of β-actin and TNF-α, interleukin-1β (IL1β), and IL6 using primers designed by SA Biosciences (Qiagen). Expression of β-actin was used to standardize the samples, and the results are expressed as a ratio relative to control.
10
Brain Tissue FITC-Albumin Quantification
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The left hemisphere of FITC-albumin injected brains were homogenized in radioimmunoprecipitation assay (RIPA) buffer (10 mmol/l Tris, 140 mmol/l NaCl, 1% Triton X-100, 1% Na dexycholate, 0.1% SDS and protease inhibitor cocktail pH 7.5) using a PowerGen 125 homogenizer (Fisher Scientific, Hampton, NH). Samples were then centrifuged for 10 min at 10,000 rpm at 4 °C and supernatants were saved. Protein concentration in the supernatant was determined using a BCA protein assay (Pierce Biotechnology, Waltham, MA) and read on a Synergy H1 Hybrid Multi-Mode Reader (BioTek, Winooski, VT). Samples were normalized for protein content, and read on a fluorescent plate reader at 488 nm excitation and 525 nm emission.
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