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L7 55

Manufactured by Beckman Coulter
Sourced in United States

The L7-55 is a centrifuge designed for laboratory applications. It can accommodate sample volumes up to 4 liters and provides controlled acceleration and deceleration for precise separations. The L7-55 features a durable stainless steel chamber and a digital display for monitoring run parameters.

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10 protocols using l7 55

1

Isolation of Membrane Protein Fractions

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Parts of frozen livers from individual animals were weighed and homogenized with a glass-Teflon homogenizer in ten volumes (w/v) of ice-cold DEA buffer (0.25% diethyl amine, 100 mM NaCl, protease and phosphatase inhibitors) and centrifuged (100,000 × g, 30 min, 4°C, Beckman L7-55, Brea, CA, United States). The pellets were homogenized in the same volume of 1% Triton buffer (1% Triton, 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 2 mM EDTA, protease and phosphatase inhibitors) with a glass-Teflon homogenizer. Homogenates were put through a 23-gauge needle, incubated for 30 min on ice, and centrifuged (100,000 × g, 30 min, 4°C, Beckman L7-55, Brea, CA, United States). The supernatants were used as membrane fractions. Protein content of all fractions was determined by the method of Spector (21 (link)) using bovine serum albumin as a standard.
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2

Isolating Cell Membranes and Lipid Rafts

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Ultracentrifugation and density gradient methods were employed to obtain total cell membranes from BeWo cells, brush border membranes from human term placenta, and lipid rafts using a Beckman L7-55 ultra-centrifuge (Beckman Coulter, Brea, CA) [27 (link)–29 (link)].
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3

Microvesicles Isolation from Bacterial Cultures

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Bacteria for MVs isolation were grown in broth culture as described in Section 4.4. Bacteria were removed by two subsequent centrifugation steps at 8000× g and 10,000× g, each for 10 min at 10 °C. The supernatant was collected and passed through a 0.45 μm pore size nitrocellulose filter (Millipore, Burlington, MA, USA). The sterility of the filtrate was assessed by a standard plate count method. The filtrates were concentrated in a Vivaspin 20 (PES membrane, 300 kDa molecular weight cut off (MWCO)) centrifugal concentration system (Sartorius, Göttingen, Germany) at 3000× g for 2 h at 10 °C. The concentrated supernatants were diluted with 20 mM Tris-HCl pH 7.4 and layered on the top of a 5 mL cushion consisting of 40% (v/v) iodixanol. The samples were centrifuged at 85,000× g for 4 h at 10 °C in a Beckman ultracentrifuge (L7-55, rotor SW28). The layer of MVs was collected from the top of the iodixanol cushion. The samples were diluted 10-fold, and ultrafiltered in the Vivaspin 20 (PES, MWCO 300 kDa) centrifugal concentrators at 3000× g for 3 h at 10 °C, with one washing step to remove residual iodixanol. MVs were diluted in Tris-HCl buffer (pH 7.4) to the final volume of 400 μL and stored at −20 °C.
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4

Bacillus thuringiensis Spore Isolation

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Ls 2362 was grown in MBS broth25 (link), and Bti strains 4Q5, 4Q7/pWF45, 4Q7/pBU-cyt1Aa-binA, and 4Q7/p45S1 were grown in 50 ml of NBG13 (link), 14 (link) appropriately supplemented with 25 μg/ml erythromycin and 3 μg/ml tetracycline, at 28 °C for 4 days by which time >95% of the cells had sporulated and lysed. Spores and crystals were collected by centrifugation at 6,500 g for 15 min, washed 2x in double-distilled (dd) H2O, followed by centrifugation at 6,500 g for 15 min at 4 °C after each wash, and lyophilized (FreezeZone 4.5, Labconco) for storage.
To isolate parasporal bodies, spore/parasporal body mixtures collected from 50 ml cultures were resuspended in 15 ml ddH2O and sonicated twice at 50% duty cycle for 15 s using the Ultrasonic Homogenizer 4710 (Cole-Parmer Instrument Co.). Five-milliliter samples were loaded onto a sucrose gradient cushion (30–65% w/v), which was then centrifuged at 20,000 g for 45 min at 20 °C in a Beckman L7–55 ultracentrifuge using the SW28 rotor. Bands containing parasporal bodies were collected and washed twice in ddH2O, followed by centrifugation at 6,500 g for 15 min at 4 °C after each wash and lyophilized for storage.
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5

Purification and Characterization of Phage

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Three mL of high-titer phage preparation were centrifuged using the preformed CsCl gradient (1.25 g/mL, 1.4 g/mL, 1.5 g/mL and 1.7 g/mL, 2.5 mL each) at 10 °C, 25,000 rpm, for 2.5 h in a Beckman Coulter ultracentrifuge, L7-55, using a SW 41 Ti rotor. Further, ten µL of the concentrated phage suspension (109 PFU/mL) were applied to carbon-coated copper grids (400 mesh) and negatively stained with 1% uranyl acetate. The grids were analyzed using a JEM 1200EX (JEOL, Tokyo Japan) transmission electron microscope at 80 kV accelerating voltage. Images were taken on Kodak film SO-163 (Kodak, Cat. \# 74144, Hatfield, PA, USA). Phage particle dimensions were measured using ImageJ version 1.53e in relation to the scale bar generated by the microscope.
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6

Lentiviral Vectors for Cell Cycle Monitoring

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Lentiviral vectors mCherry-hCdt1(30/120)/pCSII-EF-MCS (DDBJ/EMBL/GenBank, AB512478) and mVenus-hGeminin(1/100)/pCSII-EF-MCS (DDBJ/EMBL/GenBank, AB512479) were purchased from the Riken Brain Science Institute, Japan (Dr. Atsushi Miyawaki, head of provider laboratory, and Dr. Hiroyuki Miyoshi, developer of pCSII-EF-MCS). Lentiviral particles were generated by co-transfection of HEK-293TN cells (System Biosciences) with mCherry-hCdt1 (30/120)/pCSII-EF-MCS or mVenus-hGeminin (1/100)/pCSII-EF-MCS lentiviral vectors, alongside the packaging plasmid psPAX2 (Addgene plasmid, #12260) and the envelope plasmid pMD2.G (Addgene plasmid, #12259). The culture supernatant was collected and concentrated by ultracentrifugation at 22,000 rpm for 3 h (Beckman L7-55 with SW32Ti rotor) at 4 °C. The virus titer was estimated by transduction on HeLa cells and subsequent flow cytometric analysis for fluorescent protein expression.
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7

Lipid Peroxidation and Antioxidant Analysis in Tadpoles

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Whole bodies of larvae were finely chopped and mixed to obtain as much homogenous material as possible. After that, 0.2 g was taken to determine the concentration of thiobarbituric acid reactive substance (TBARS) as a standard marker for LPO (lipid peroxidation); the rest was used for determination of antioxidant parameters. Tadpoles were individually homogenized with an Ultra Turrax homogenizer T-18 (IKA-Werk, Staufen, Germany) at a 1:5 ratio in an ice cold 25 mM sucrose buffer (pH 7.4) containing 10 mM Tris-HCl and 5 mM EDTA. The homogenates were sonicated with an ultrasonic homogenizer (Sonopuls HD 2070, Bandelin electronic, Berlin, Germany) for 30 s at 10 kHz. A part of the sonicate was taken for measurement of total reduced glutathione (GSH) concentration, while the rest was centrifuged in an L7-55 ultracentrifuge (Beckman, Brea, CA, USA) at 100,000× g at 4 °C for 90 min [32 (link)]. The supernatants were used for measuring AOS parameters.
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8

Transmission Electron Microscopy of Phage

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For transmission electron microscopy (TEM) analysis, the high-titer phage preparation underwent CsCl density gradient centrifugation. In this procedure, two mL of the phage preparation was meticulously layered onto a preformed CsCl gradient with specific densities of 1.3 g/mL, 1.4 g/mL, 1.5 g/mL, 1.6 g/mL, and 1.7 g/mL, each occupying 2 mL of the gradient. Ultracentrifugation was carried out using a Beckman Coulter L7-55 ultracentrifuge equipped with an SW 40 Ti rotor. The centrifugation process was conducted at a temperature of 18 °C, with a centrifugal force of 110,961.5 g for a duration of 1.5 h.
Following the density gradient centrifugation, five microliters of the phage suspension was loaded onto 400 mesh carbon-formvar-coated copper grids and negatively stained with 1% uranyl acetate. Phage particles were visualized using a JEM-100C transmission electron microscope (JEOL, Akishima, Japan) with an accelerating voltage of 80 kV. Images were taken on Kodak film SO-163 (Kodak, Cat. #74144, Hatfield, PA, USA) with 45,000× magnification. Phage particle dimensions were measured for 10 phage particles using ImageJ version 1.53e [56 (link)] in relation to the scale bar generated from the microscope.
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9

Plant Extract Preparation for Biochemical Analysis

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Thoroughly washed plants were finely cut or chopped using a mechanical blender, and soaked in an equal quantity of M63 medium supplemented with 0.4% DIECA. Plant debris was removed by filtration and subsequent centrifugation at 85,000× g for 0.5 h at 4 °C in a Beckman ultracentrifuge (L7-55, rotor SW28). The extract was then sterilised by filtration through a 0.22 μm nitrocellulose filter (Millipore, Burlington, MA, USA) and stored at −80 °C for further use.
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10

Donkey Milk Fractionation Protocol

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Milk samples were kept at room temperature (20°C) for 3 h before the first step of the separation process, occurred within 15 h from collection. Whole milk samples (n = 16) were centrifuged at 1,000 × g, 20 min at room temperature to separate fat and to obtain skim milk as described by Uniacke-Lowe et al. (2013) (link). Skim milk samples were ultracentrifuged (rotor 50 Ti, Beckman L7-55 centrifuge, Beckman Coulter, Brea, CA) at 100,000 × g for 60 min at 4°C to separate a sedimentable casein pellet and to obtain a supernatant whey (soluble) fraction (Anastácio et al., 2004) (link), which was then ultrafiltered (Amicon, Ultra-4, cut-off 3kDa; Sigma-Aldrich, Milano, Italy) at 7,500 × g for 60 min at room temperature to obtain the aqueous phase of donkey milk. Aliquots of whole milk, skimmed, ultracentrifuged, and ultrafiltered milk fractions were frozen at -21°C until analysis.
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