Mice in experimental and control groups were anesthetized with 0.02 mL/g of Avertin. Once mice were anesthetized, a glucose reading was taken using Accu-Check Nano meter (140–200 mg/dL). Mice were then injected intraperitoneally with epinephrine (0.75 ug/g, dissolved in normal saline), propranolol (0.04 mg/g, dissolved in normal saline), glucagon (1 ug/g, dissolved in 0.05% acetic acid) or saline. Approximately 30 min after the injection, another glucose reading was taken and recorded (~150–250 mg/dL for controls and ~300–500 mg/dL for experimental). Animals were subjected to cervical dislocation, dissected, and tissues were removed and put directly into liquid nitrogen. Frozen tissues were then crushed into powder using tissue pulverizer (Cellcrusher.com). The resulting powdered tissues were weighed, 100 uL/0.01 g lysis buffer (50 mM Tris-HCL, 100 mM NaF, 10 mM EDTA, 2 mM EGTA, 10 mM BetaGP, 1x Protease inhibitor) was added, and then subjected to Polytron (Cat#97057–700 VWR) tissue disruption. Resulting lysates were spun 2 × 10,500 rpm for 10 min. Protein concentration was determine using protein assay dye (Bio-Rad #5000006). 20 ul of 5x SDS lysis buffer was added to 80 ul of lysate and then westerns were performed probing for pS6K, S6K, pCREB, and CREB.
Polytron
Manufactured by Avantor
Sourced in United States
The Polytron is a high-performance laboratory homogenizer designed for the effective dispersion and homogenization of samples. It utilizes a powerful motor and specialized dispersing tool to thoroughly mix and disintegrate a wide range of materials, including tissues, suspensions, and emulsions.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Protein assay dye, by Bio-Rad (1 mentions)
Tri reagent rna isolation reagent, by Merck Group (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Xf96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
5 protocols using polytron
1
Glucose Modulation and Protein Analysis in Mice
2
Pancreatic RNA Purification Protocol
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For gene expression profiling, we implemented a method for purification of intact pancreatic RNA according to a modified guanidinium salts method (MacDonald et al, 1987 ). Briefly, a small piece of pancreatic tissue was immediately homogenized with a Polytron (VWR) in pre‐cooled 4 M guanidinium thiocyanate with 112 mM beta‐mercaptoethanol, centrifuged at 5,000 g for 5 min at 4°C to remove insoluble debris. RNA was precipitated from the supernatant with pre‐cooled 75% ethanol, 0.1M potassium acetate, pH 5.5, and 75 mM acetic acid at −20°C for 2 h. The precipitate was pelleted by centrifugation at 10,000 g for 10 min at 4°C and resuspended at room temperature in 7.5 M guanidinium HCl and 10.5 mM beta‐mercaptoethanol. The RNA was re‐precipitated twice with 0.1 M potassium acetate, pH 5.5, and 50% ethanol to remove residual RNases, followed by purification with TRI Reagent RNA Isolation Reagent (Sigma‐Aldrich). The concentration and quality of RNA was measured with a NanoDrop spectrophotometer (ND‐1000, Thermo Scientific) and an Agilent 2100 Bioanalyzer. RNA integrity numbers ranged from 7.8 to 9.3.
Alternatively, we used a previously described protocol (Cobo et al,2018 ).
Alternatively, we used a previously described protocol (Cobo et al,
3
Quantifying mRNA Expression in Brain Tissue
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Trizol was used
for the extraction of the RNA by homogenizing the brain tissue using
a polytron (VWR) device. A reverse transcription kit was used to transcribe
the RNA samples into cDNA. Real-time PCR was used under the following
conditions: 40 cycles at 95 °C for 5 min. Primer sequences are
given inTable 4 . Different
markers of mRNA expression were analyzed by real-time PCR GAPDH as
a reference using green qPCR master mix plus for assay. The PCR products’
mRNA expressions were quantified.67 (link)
for the extraction of the RNA by homogenizing the brain tissue using
a polytron (VWR) device. A reverse transcription kit was used to transcribe
the RNA samples into cDNA. Real-time PCR was used under the following
conditions: 40 cycles at 95 °C for 5 min. Primer sequences are
given in
markers of mRNA expression were analyzed by real-time PCR GAPDH as
a reference using green qPCR master mix plus for assay. The PCR products’
mRNA expressions were quantified.67 (link)
4
Quantitative Real-Time PCR Analysis of Gene Expression
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RNA was extracted
from rat brain hemispheres. The brain tissue was homogenized using
a Polytron (VWR) device and then treated with Trizol (Life Technologies,
Carlsbad, CA, USA). RNA samples were transcribed to cDNA in a 20 μL
volume using the QuantiTect reverse transcription kit (Qiagen).
The thermal cycling comprised the real-time PCR as per the following
conditions: 95 °C for 5 min followed by 40 cycles (denaturation
for 15 s at 95 °C, annealing for 20 s at 60 °C, and extension
for 20 s at 72 °C). The primer sequences and PCR product size
for the target and reference genes are listed inTable 6 .
mRNA expression levels of different markers were detected
by real-time
PCR with GADPH as an internal reference, using a Mesa Blue qPCR Master
Mix Plus for the SYBR assay (Eurogentec) on the Master cycler Realplex2
(Eppendorf).
Relative quantitation was calculated by the comparative
threshold
cycle (CT) method with realplex software.
Mean CT of triplicate measurements was
used to calculate ΔCT as the difference
in CT for target and internal reference
(GADPH) genes. The difference between the ΔCT of the control experiment and the ΔCT of each sample was calculated to give ΔΔCT. Fold increase in mRNA was calculated by 2–ΔΔCT.
The PCR products of tissue samples after real-time PCR were electrophoresed
by E-Gel Precast Agarose Electrophoresis System.
from rat brain hemispheres. The brain tissue was homogenized using
a Polytron (VWR) device and then treated with Trizol (Life Technologies,
Carlsbad, CA, USA). RNA samples were transcribed to cDNA in a 20 μL
volume using the QuantiTect reverse transcription kit (Qiagen).
The thermal cycling comprised the real-time PCR as per the following
conditions: 95 °C for 5 min followed by 40 cycles (denaturation
for 15 s at 95 °C, annealing for 20 s at 60 °C, and extension
for 20 s at 72 °C). The primer sequences and PCR product size
for the target and reference genes are listed in
mRNA expression levels of different markers were detected
by real-time
PCR with GADPH as an internal reference, using a Mesa Blue qPCR Master
Mix Plus for the SYBR assay (Eurogentec) on the Master cycler Realplex2
(Eppendorf).
Relative quantitation was calculated by the comparative
threshold
cycle (CT) method with realplex software.
Mean CT of triplicate measurements was
used to calculate ΔCT as the difference
in CT for target and internal reference
(GADPH) genes. The difference between the ΔCT of the control experiment and the ΔCT of each sample was calculated to give ΔΔCT. Fold increase in mRNA was calculated by 2–ΔΔCT.
The PCR products of tissue samples after real-time PCR were electrophoresed
by E-Gel Precast Agarose Electrophoresis System.
5
Mitochondrial Function Assay Protocol
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Mitochondria were isolated from heart in MSHE (70-mM sucrose, 220-mM mannitol, 10-mM Hepes, 1-mM EGTA, and 0.5% BSA, pH 7.2). Tissue was minced with a tissue homogenizer (Polytron; VWR) and centrifuged twice at 800 g to remove cellular debris. The mitochondrial fraction was isolated with an 8,500 g spin, and the pellet was resuspended and spun a second time. The final pellet was resuspended in 50 µl MSHE, and protein quantification was performed. Mitochondria were diluted in MAS/PM (70-mM sucrose, 220-mM mannitol, 10-mM Hepes, 1-mM EGTA, 0.2% BSA, 10-mM KH2PO4, 5-mM MgCl2, pH 7.2, 5-mM pyruvate, and 2.5-mM malate), plated at 0.8 µg/well, and analyzed on the XF96 Extracellular Flux Analyzer (Seahorse Bioscience). Drugs added were 4-mM ADP, 5-µM oligomycin, 5-µM CCCP, and 5-µM antimycin A.
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