Genomic yeast DNA was isolated as described by Graham et al. [23 (link)], with some modifications. Yeast cells were separated from the TSB medium by centrifugation at 14,000× g for 7 min. Each pellet was washed with sterile de-ionized water and centrifuged at 14,000× g for 7 min. Then, the pellet was suspended in a 600 μL extraction buffer (1 M Tris pH 8.0, 10% sodium dodecyl sulfate) (Sigma-Aldrich, Poznan, Poland) and incubated at 100 °C for 30 min in a water bath. The lysate was centrifuged at 14,000× g for 7 min, and after centrifugation the supernatant was put into a new tube and phenolated three times. A mixture of phenol/chloroform/isoamyl alcohol (25:24:1; v/v/v) (Sigma-Aldrich, Poznan, Poland) was used in two first phenolates, and a mixture of chloroform/isoamyl alcohol (24:1; v/v) was used in the last phenolate. Then, DNA was extracted with 1/10 estimated sample volumes of 7.5 M sodium acetate (pH 5.2) (Sigma-Aldrich, Poznan, Poland), and two estimated sample volumes of 98% ethanol (Sigma-Aldrich, Poznan, Poland) at −20 °C for 1 h, and centrifuged at 14,000× g for 17 min. The extracted DNA was washed twice with 70% ethanol and centrifuged each time at 14,000× g for 7 min. The extract was dried at 37 °C for 30 min in an incubator. The dried pellet was dissolved in 100 μL of sterile de-ionized water at 37 °C for 20–24 h and was stored at −20 °C for later study.
Extraction buffer
Manufactured by Merck Group
Sourced in Poland, United States, Germany
Extraction buffer is a laboratory reagent used to prepare samples for analysis. It is designed to facilitate the extraction and solubilization of target analytes from various types of samples, such as biological materials or environmental samples. The core function of the extraction buffer is to create an optimal chemical environment for the extraction process, enabling the efficient recovery of the analytes of interest. The specific composition and properties of the extraction buffer may vary depending on the application and the nature of the samples being analyzed.
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Lab products found in correlation
Glucose uptake colorimetric assay kit, by Merck Group (2 mentions)
Neutralization buffer, by Merck Group (2 mentions)
Sodium acetate, by Merck Group (1 mentions)
Phenol chloroform isoamyl alcohol, by Merck Group (1 mentions)
Chloroform isoamyl alcohol, by Merck Group (1 mentions)
Electrochemiluminescence solution, by PerkinElmer (1 mentions)
Optiprep, by Merck Group (1 mentions)
Nuclei pure storage buffer, by Merck Group (1 mentions)
Nadp nadph quantification kit, by Merck Group (1 mentions)
Nad nadh quantification kit, by Merck Group (1 mentions)
Precellys evolution homogenizer, by Bertin Technologies (1 mentions)
Polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ripa lysis, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Sds polyacrylamide gel, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Tween 20, by Merck Group (1 mentions)
Tris buffered saline (tbs), by Merck Group (1 mentions)
11 protocols using extraction buffer
1
Genomic Yeast DNA Extraction
2
Protein Extraction and Detection Protocol
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Extraction buffer (radioimmunoprecipitation assay buffer plus 1× proteinase and phosphatase inhibitors; Sigma-Aldrich, St. Louis, MO, USA) was used to extract the total protein content from 200 mg powders or 1 × 106 cells. After the sample was placed in 10% resolving 1× sodium dodecyl sulfate–polyacrylamide gel, the proteins were transferred to a nylon membrane. Blocking was achieved by placing the nylon membrane in 5% skimmed milk in 1× phosphate-buffered saline containing 0.05% Tween 20 (1× PBST); subsequently, primary and secondary antibodies were diluted in 2% skimmed milk in 1× PBST (Supplementary Table S1 ) for protein detection. Signals were developed by using electrochemiluminescence solution (PerkinElmer, Waltham, MA, USA) and films (GE Healthcare, Chicago, IL, USA).
3
Glucose Uptake Measurement in Apoptotic Cells
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LR73 cells were incubated with apoptotic Jurkat cells for 2h, washed 3 times with PBS and incubated with 10 mM 2-deoxygloucs (2-DG), a glucose analog, in glucose free media for 30 min. Following incubation, cells were washed 3 times with PBS and lysed with Extraction Buffer (Sigma Cat#: MAK083). Lysate was frozen/thawed in dry ice/ethanol, and then heated at 85°C for 40 min. Lysate was then cooled on ice for 5 min and then neutralized by Neutralization Buffer (Sigma Cat#: MAK083). Samples were spun down at 13,000x g to remove insoluble fraction and then diluted 10-fold by adding Assay Buffer. Using the lysate, glucose uptake was measured using Glucose Uptake Colorimetric Assay Kit (Sigma). 2-DG is taken up by cells and phosphorylated by hexokinase to 2-DG6P. 2-DG6P cannot be further metabolized and accumulates in cells, directly proportional to the glucose uptake by cells. 2-DG uptake is determined by a coupled enzymatic assay in which the 2-DG6P is oxidized, resulting in the generation of NADPH, which is then determined by a recycling amplification reaction in which the NADPH is utilized by glutathione reductase in a coupled enzymatic reaction that produces glutathione. Glutathione reacts with DTNB to produce TNB, which was detected at 412 nm as per the manufacturer’s recommendations.
4
Migrasome Enrichment via Gradient
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Cell fractionation was performed by Iodixanol-sucrose density gradient centrifugation, using Optiprep (Sigma-Aldrich, D1556). Cells were collected and lysed in extraction buffer (Sigma-Aldrich, E1156), then the samples were centrifuged at 1000× g for 10 min and then at 2000× g for 20 min. The supernatant, containing the crude migrasome fraction, was fractionated at 150000× g for 4 h in a multi-step Optiprep dilution gradient. The gradient was: 2%, 5%, 8%, 10%, 12%, 15%, sample (19%), 25%, 30%. Fractions are prepared for TEM observation to determine fractions in which PLSs are enriched.
5
Exploring Gut and Immune Responses in Rat Swimming Exercise
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After 7 weeks of swimming exercise, rats were fasted for 4 h. Samples of blood was centrifuged for 15 min at 1, 000 × g, and serum samples were harvested and stored at -20°C for detection of LDH release and inflammatory mediators. At the time of euthanasia, feces were collected and stool samples were extracted in the extraction buffer (Sigma), centrifuged for 5 min at 13,000 × g, and then used for the measurement of calprotectin and lactoferrin. After all animals were euthanized, laparotomy was performed immediately. The spleen was dissected for photoing and weighing. The colon was removed from the distal end of the cecum to the rectum, freed of adherent adipose tissue, split longitudinally, and washed with ice-cold saline to remove fecal residue and photographed. Isolated colons were utilized for histopathological, immunohistochemical, biochemical investigation, and other analyses.
6
Isolation of Nuclear Fractions from Stroke-Affected Mouse Brains
7
Quantifying NADP(H) and NAD(H) Levels
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Enzymatic cycling assays to determine NADP(H) and NAD(H) levels were performed according to the NADP+/NADPH Quantification Kit (Sigma) and NAD+/NADH Quantification Kit (Sigma). Cells were grown in Delft + 100 mg L−1 histidine + 100 mg L−1 uracil medium, then 5 OD600nm of cells were collected at later exponential (EX) phase (OD600nm ≈ 2) and post-diauxic (PD) phases. Collected cells were immediately added to 20 ml of cold methanol (pre-chilled to −80 °C) to quench cellular metabolism. Cells were centrifuged at −10 °C for 4 min (4000×g), then supernatant was removed. Cell pellets were freeze-dried for 2 h and stored at −80 °C until measurement. The dried cell pellet was resuspended in 500 µl of extraction buffer (Sigma) and added to 0.2 g of prechilled glass beads (425–600 µm diameter, Sigma). Cells were lysed via four rounds of vortexing for 20 s at speed 6200 rpm on the Precellys Evolution Homogenizer (Bertin technologies) followed by incubation for 1 min on ice. The cell lysates were centrifuged at 14000×g for 1 min at 0 °C, and supernatant was used to measure NADH(H) and NAD(H) amounts following the manufacturer’s instructions (Sigma).
8
Western Blot Protein Detection
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After extraction using RIPA lysis (Thermo Scientific) and extraction buffer (Sigma Aldrich) with 100X anti‐protease and anti‐phosphatase, the proteins were separated on a 15% Tris‐Glycine buffer Polyacrylamide gel (Thermo Fisher) and transferred to nitrocellulose membrane (Sigma Aldrich). The membranes were saturated for 1 h in 5% (w/v) TBS Tween 0.1% no‐fat milk. The membranes were incubated overnight at 4°C with the primary antibody solution in 5% (w/v) TBS Tween 0.1% no‐fat milk (detailed antibodies in Table S4 ). Membranes were then incubated with the secondary antibody solution in 5% (w/v) TBS Tween 0.1% no‐fat milk for 1 h at room temperature. After several washes with 0.1% TBS Tween, blots were visualized using chemiluminescent reagents (Super Signal Pico, Thermo Fisher) on the ChemiDoc Universal HoodII device (Bio‐rad).
9
Glucose Uptake Measurement in Apoptotic Cells
10
Extraction and Loading of Sulforaphane in Biomimetic Vesicles
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The leaves were cut into small pieces before vacuum filtering with an extraction buffer (0.5 M sucrose, 1 mM DTT, 50 mM HEPES and 1.37 mM ascorbic acid at pH 7.5) (Sigma-Aldrich, Darmstadt, Germany) supplemented with 0.6% PVP. The mixture was homogenized using a blender and filtered through a nylon mesh (pore diameter of 100 µm). The filtrate was centrifuged (10,000× g, 30 min, 4 °C). The supernatant was recovered and ultracentrifuged (100,000× g, 35 min, 4 °C), and the pellet obtained was suspended in 500 µL of FAB buffer (5 mM potassium phosphate buffer and 0.25 M sucrose, pH 6.5) and stored at −80 °C until their later use. The protein concentration in the isolated microsomal fraction was determined using the Bradford method [74 (link)] with bovine serum albumin (BSA) as the standard. To obtain SFN-loaded BM-vesicles, the drug, at a concentration of 500 µM dissolved in FAB buffer, was mixed with BM-vesicles (0.2 protein mg mL−1) with vigorous shaking, reaching a total volume of 2 mL.
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