Liver pieces were weighed and homogenized using a dounce homogenizer (Kimble Chase) in 500 µl 80% methanol including an internal metformin standard at a final concentration of 2(µg/ml, diluted from Metformin-d6 hydrochloride stock (Santa Cruz, 1mg resuspended in 1 ml DMSO). Samples were diluted prior to analysis as needed to ensure that measurements were within the linear range of measurements determined by a standard curve. Metformin and the internal standard (metformin-d6) were analyzed on an Agilent 6410 triple quadrupole mass spectrometer in MRM mode, coupled to an Agilent 1100 lc system. The transition states monitored were 136.1 −> 60.1 (quantitative transition), and 60.1 (qualitative transition) for the internal standard, and 130 −> 60.1 (quantitative) and 71.1 (qualitative) for metformin. Spray chamber settings were: N2 drying gas flow = 10L/min, drying gas temp = 350°C, and nebulizer pressure = 30psi. Capillary voltage was set to 4000V. Column used was a phenomenex 2.0 ×50mm Kinetex C18. Mobile phase A = H2O/40mM heptafluorobutyric acid (HFBA), and mobile phase B=MeOH. Flowrate was 200 µl/min. Gradient was T=0 90:10 to T=5min 5:95, with stop time at 10 minutes, followed by a 4 minute re-equilibration step (post-time). 5 µl was injected. Concentrations were calculated from a 5-point calibration curve. The concentration of metformin was normalized to tissue weight.
Dounce homogenizer
Manufactured by DWK Life Sciences
Sourced in United States
The Dounce homogenizer is a manual laboratory device used to gently disrupt cells or tissues for the extraction of intracellular components. It consists of a glass or Teflon pestle that fits snugly within a matching glass or Teflon vessel, allowing for controlled mechanical disruption of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Nuclei ez lysis buffer, by Merck Group (9 mentions)
Am2696, by Thermo Fisher Scientific (8 mentions)
N2615, by Thermo Fisher Scientific (8 mentions)
Protease inhibitor, by Roche (5 mentions)
Nuclei buffer, by Genomics Plc (4 mentions)
Phrodo green, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by DWK Life Sciences (2 mentions)
H3k9me3, by Merck Group (2 mentions)
Bioruptor, by Diagenode (2 mentions)
6410 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
1100 lc system, by Agilent Technologies (1 mentions)
Sw41 rotor, by Beckman Coulter (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared fluorescent imaging system, by LI COR (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Xcell surelock system, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
21 protocols using dounce homogenizer
1
Metformin Quantification in Liver Tissue
2
Single Nucleus Isolation from Mouse Kidney
3
Subcellular Fractionation of TM-3 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Subcellular fractionation was performed according to the method described in Ding et al. (2013) (link) and Gong et al. (2011) (link) with slight modifications. TM-3 cells were harvested, washed with PBS, and resuspended in hypotonic buffer (10 mM Hepes, pH 7.8, 1 mM EGTA, and 25 mM KCl) for 20 min on ice. Cells were spun down at 600 g for 5 min, and the supernatants were discarded. Cells were resuspended with TES buffer (20 mM Tris, pH 7.4, 1 mM EDTA, and 250 mM sucrose) and homogenized through Dounce homogenizer (Kimble Chase) with loose pestle 25 times. The postnuclear supernatants (PNSs) were obtained after centrifugation at 1,000 g for 10min. PNS was further centrifuged at 12,000 g for 15 min to get postmitochondrial fraction. Floating buffer (100 mM NaCl, 20 mM Tris, pH 7.4, and 1.5 mM MgCl2) was laid on postmitochondrial fraction and centrifuged at 200,000 g for 1 h using a Beckmann SW41 rotor. The pellet fraction and middle gradient were collected as crude microsomal fraction and cytosol, respectively. The LDs on the top were collected and washed three times with floating buffer. All procedures were performed at 4°C, and protease inhibitors were added. Protein concentration was determined using BCA assay (Thermo Fisher Scientific). Relative amounts of protein on each fraction were analyzed by Western blot.
4
Dissection and Fractionation of Hippocampal Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
P21 Balb/cByj pups raised in the vivarium were anesthetized with chloral hydrate (100 mg/kg) and the hippocampus was dissected out and placed in a 2-ml Dounce homogenizer (Kimble Chase) containing 500 μl of ice-cold buffer A [HEPES 4 mM pH 7.4, 320 mM sucrose, 1 mM DTT, and protease inhibitor cocktail (cat. No. P2714, Sigma)] and homogenized sequentially using plunger A (5 S) and plunger B (5 S). The homogenate was transferred into a cold Eppendorf tube and centrifuged for 5 min at 1000 g at 4 °C to remove nuclei and cell debris (P1 fraction). The supernatant was transferred to a new Eppendorf tube and centrifuged for 20 min, 15,000g, at 4 °C. The supernatant containing soluble proteins was transferred to a new Eppendorf tube (S2) and snap frozen in liquid nitrogen. The remaining pellet (P2 fraction), containing the crude synaptosomes was stained with pHrodo green (Thermo Fisher P36013) using 1:10 protein:dye molar ratio and resuspended at 1 mg/ml concentration in 5 % DMSO in buffer A and frozen at −80 °C freezer until further used for phagocytosis assay (Fig. S4 ).
5
Dissection and Fractionation of Hippocampal Tissue
6
Nuclei Isolation for snATAC-seq and snRNA-seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For snATAC-seq, nuclei were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (5892791001; Roche). Samples were cut into <2 mm pieces, homogenized using a Dounce homogenizer (885302–0002; Kimble Chase) in 2 ml of ice-cold Nuclei EZ Lysis buffer, and incubated on ice for 5 min with an additional 2 ml of lysis buffer. The homogenate was filtered through a 40 μm cell strainer (43–50040–51; pluriSelect) and centrifuged at 500 × g for 5 min at 4 °C. The pellet was resuspended, washed with 4 ml of buffer, and incubated on ice for 5 min. Following centrifugation, the pellet was resuspended in Nuclei Buffer (10× Genomics, PN-2000153), filtered through a 5 μm cell strainer (43-50005-03, pluriSelect), and counted. For snRNA-seq preparation, the RNase inhibitors (Promega, N2615 and Life Technologies, AM2696) were added to the lysis buffer, and the pellet was ultimately resuspended in nuclei suspension buffer (1× PBS, 1% bovine serum albumin, 0.1% RNase inhibitor). Subsequently, 10X Chromium libraries were prepared according to manufacturer protocol.
7
Western Blot Analysis of Smad1 in Cortical Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates from freshly collected cortical tissue were prepared in RIPA buffer (Sigma), supplemented with protease and phosphatase inhibitors (Sigma), and homogenized using a Dounce homogenizer (Kimble Chase). Protein levels were quantified using BCA assays (Pierce Thermo Scientific). Proteins were resolved on NuPAGE gels (Invitrogen) using the XCell SureLock system (Invitrogen). The immunoreactive bands were detected by fluorescent ODYSSEY infrared imaging system (LI-COR). Equal protein loading was controlled by probing the blots with an antibody to β-actin. Antibodies used were rabbit anti-Smad1 (Abcam, 1:1000) and mouse anti-β-actin (Thermo Scientific, 1:2000).
8
Nuclei Isolation for snATAC-seq and snRNA-seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For snATAC-seq, nuclei were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (5892791001; Roche). Samples were cut into < 2 mm pieces, homogenized using a Dounce homogenizer (885302-0002; Kimble Chase) in 2 ml of ice-cold Nuclei EZ Lysis buffer, and incubated on ice for 5 min with an additional 2 ml of lysis buffer. The homogenate was filtered through a 40-μm cell strainer (43-50040-51; pluriSelect) and centrifuged at 500g for 5 min at 4 °C. The pellet was resuspended, washed with 4 ml of buffer, and incubated on ice for 5 min. Following centrifugation, the pellet was resuspended in Nuclei Buffer (10× Genomics, PN-2000153), filtered through a 5-μm cell strainer (43-50005-03, pluriSelect), and counted. For snRNA-seq preparation, the RNase inhibitors (Promega, N2615 and Life Technologies, AM2696) were added to the lysis buffer, and the pellet was ultimately resuspended in nuclei suspension buffer (1× PBS, 1% bovine serum albumin, 0.1% RNase inhibitor)65 (link). 10X Chromium libraries were prepared according to manufacturer protocol.
9
ChIP-seq Protocol for Testis Tissue Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To perform ChIP, small pieces of frozen testis tissue were cross-linked with 2% (w/v) formaldehyde at room temperature for 30 min using end-over-end tumbler. We next crushed fixed tissues in the presence of ChIP lysis buffer (1% (w/v) SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) by 40 strokes with a B pestle in a Dounce homogenizer (Kimble-Chase, Vineland, USA). Thereafter, lysate was sonicated using Covaris ultrasonicator (Covaris, E220) to shear the chromatin to 150–200 bp. Lysate was then diluted 1:10 with ChIP dilution buffer (0.01% (w/v) SDS, 1.1% (w/v) Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). We performed immunoprecipitation using 6 µg rabbit polyclonal anti-H3K27Ac antibody (Abcam, ab4729). Following the immunoprecipitation, we extracted DNA with phenol:chloroform:isoamyl alcohol (25:24:1; pH 8) and prepared libraries for anti-H3K27ac, and input DNA as previously described68 (link). ChIP-seq libraries were sequenced as 79-nt paired-end reads using NextSeq500 (Illumina).
10
ChIP Analysis of Plasmodium falciparum Epigenetics
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!