The THP-1 monocytes were seeded at a concentration of 6x105 cells/mL in 75 cm2 flasks and differentiated in M(0) THP-1. Then, cells were washed with micro-filtered (0.2 μm pore size) 1X PBS w/o Ca2+ and Mg2+ and incubated with 0.0125% DMSO (Control) or 3 μM PBDE-47 in RPMI medium with 10% FBS previously depleted of microvesicles by centrifugation at 118,000 g at 4°C overnight using a Beckman SW28 rotor. Thereafter, the M(0) THP-1 macrophages were differentiated in M(LPS) THP-1 for 24 hours at 37°C in 5% CO2. The supernatants containing DMSO derived (sEVsDMSO) and PBDE derived (sEVsPBDE) small extracellular vesicles were first centrifuged at low speed to remove cells and debris. The large EVs were then isolated by centrifugation at 10,000 x g for 30 minutes at 4°C using an Eppendorf rotor F34-6-38 and resuspended in a proper volume of 1X PBS w/o Ca2+ and Mg2+. Afterwards, sEVs were collected from the supernatants into Beckman Coulter polypropylene open top tubes via centrifugation at 118,000 g for 70 minutes at 4°C using a Beckman SW28 rotor. Finally, the sEVs were washed in 1X PBS w/o Ca2+ and Mg2+ and resuspended in the same buffer.
F 34 6 38
Manufactured by Eppendorf
Sourced in Germany
The F-34-6-38 is a laboratory equipment product manufactured by Eppendorf. It is a device designed for specific laboratory functions. The core function of this product is to provide a precise and controlled environment for various laboratory applications. No further details or interpretations about the intended use of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Polypropylene open top tubes, by Beckman Coulter (3 mentions)
Sw28 rotor, by Beckman Coulter (2 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
P50at2 716, by Hitachi (1 mentions)
Potter elvehjem ptfe, by Merck Group (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
E coli bl21 de3, by Thermo Fisher Scientific (1 mentions)
Centrifuge 5810r, by Eppendorf (1 mentions)
Restriction enzyme, by Takara Bio (1 mentions)
Dna marker, by Takara Bio (1 mentions)
Protein molecular weight marker, by Takara Bio (1 mentions)
Gel extraction kit, by Omega Bio-Tek (1 mentions)
Pmd19 t vector, by Takara Bio (1 mentions)
Plasmid mini kit 1, by Omega Bio-Tek (1 mentions)
Optima l 100 xp preparative ultracentrifuge, by Beckman Coulter (1 mentions)
7 protocols using f 34 6 38
1
Isolation and Characterization of Small Extracellular Vesicles from PBDE-Treated THP-1 Macrophages
2
Extraction and Purification of Luciferase from N. gardneri Mycelium
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Mycelium of the basidiomycete fungus N. gardneri (MycoBank MB519818) was obtained from mushrooms collected in the Brazilian Coconut Forest in Altos, Piauí, Brazil (16 (link)). The mycelium was inoculated on sterilized dialysis cellulose membrane squares (3 × 3 cm, Sigma-Aldrich) in 100-mm petri dishes over a medium composed of 1.0% (w/v) sugar cane molasses (82.2° Bx, Pol 56%) and 0.10% (w/v) yeast extract (Oxoid) in 2.0% (w/v) agar (Oxoid) (14 (link)). Cultures were maintained for 14 days in darkness inside a KMF 240 climatic chamber (Binder) at 25°C and ~80% humidity. Crude extracts were typically prepared by homogenization of 2 g of mycelium in 10 ml of 100 mM phosphate buffer (pH 7.5) containing 2 μl of 1 mM phenylmethylsulfonyl fluoride as protease inhibitor (Sigma-Aldrich) at 0°C (Potter-Elvehjem PTFE, Sigma-Aldrich). After centrifugation for 10 min at 10,000g and 4°C (5804R, rotor F34-6-38; Eppendorf) to clear debris, the resulting supernatant was further ultracentrifuged for 90 min at 200,000g and 4°C (RP50T-2, rotor P50AT2-716; Hitachi). The luciferase-enriched fraction was reconstituted in 1 ml of 100 mM phosphate buffer (pH 7.5) containing 2% glycine and 1 mM dithiothreitol (DTT).
3
Fungal Anaerobic Growth Assay
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Routinely passaged A. robustus, C. churrovis, N. californiae, or P. finnis was inoculated into 60 mL serum bottles (VWR International) containing 40 mL of anaerobic, autoclaved Medium C (67 ) with 0.4 g of 4 mm-milled reed canary grass. After 3 d of growth at 39 °C, these seed cultures were used to inoculate four replicate Hungate tubes per fungal strain containing 9 mL of anaerobic, autoclaved Medium C and 0.1 g of 4 mm-milled reed canary grass. For each serum bottle or Hungate tube, 1.0 mL of fungal inoculum was used. Four Hungate tubes containing anaerobic, autoclaved Medium C and reed canary grass were incubated at 39 °C for use as a control. Cultures and controls were harvested after 6 d of incubation, centrifuged at 3,220 g for 10 min with the swinging bucket rotor (Eppendorf F-34-6-38) at 4 °C, and the fungal supernatant was frozen at −80 °C for exometabolomics analysis.
4
Heterologous Expression of Melittin Peptide
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Escherichia
coli JM109 (DE3) and E. coli BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA). Commercial
melittin (cMET, 98%) was purchased from GenScript Co., Ltd. (Nanjing,
China). PrimeStar Max (Premix), rTaq DNA polymerase,
restriction enzymes, T4 DNA ligase, DNA marker, protein molecular
weight marker, and pMD19-T vector were purchased from Takara (Dalian,
China). Primers and the melittin (MET) gene were synthesized by Shanghai
Sangon Biotechnology Co. (Shanghai, China). Plasmid Mini kit I and
the gel extraction kit were purchased from Omega Bio-Tek (USA). Expression
vector pGEX-6p-1 was purchased from Wuhan Miaoling Biological Technology
Co., Ltd. (Wuhan, China). The type of centrifuge was Eppendorf Centrifuge
5810 R, and the rotor was F-34-6-38 (Germany).
coli JM109 (DE3) and E. coli BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA). Commercial
melittin (cMET, 98%) was purchased from GenScript Co., Ltd. (Nanjing,
China). PrimeStar Max (Premix), rTaq DNA polymerase,
restriction enzymes, T4 DNA ligase, DNA marker, protein molecular
weight marker, and pMD19-T vector were purchased from Takara (Dalian,
China). Primers and the melittin (MET) gene were synthesized by Shanghai
Sangon Biotechnology Co. (Shanghai, China). Plasmid Mini kit I and
the gel extraction kit were purchased from Omega Bio-Tek (USA). Expression
vector pGEX-6p-1 was purchased from Wuhan Miaoling Biological Technology
Co., Ltd. (Wuhan, China). The type of centrifuge was Eppendorf Centrifuge
5810 R, and the rotor was F-34-6-38 (Germany).
5
Isolation of Microalgae-Derived Extracellular Vesicles
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The 50 ml batch‐cultures were centrifuged on day 30 at low speed to separate cells from the culture medium. Then, the separation of microalgae‐derived EVs was performed by differential centrifugation (dUC) (Romancino et al., 2013 (link)). Large EVs (lEVs) were isolated in 50 ml Eppendorf polypropylene conical tubes at 10,000 × g for 30 min at 4°C using an Eppendorf rotor F34‐6‐38. Small EV (sEVs) were then collected from the supernatant into Beckman Coulter polypropylene open top tubes via centrifugation at 118,000 × g for 70 min at 4°C using a Beckman SW28 rotor. After a Phosphate‐buffered saline (PBS 1X, without Calcium and magnesium, Thermo Fisher Scientific) washing step, the pellet was re‐suspended in PBS for subsequent analyses, these sEV preparations are the dUC‐isolated nanoalgosomes.
6
Ribosome Isolation from Yeast Cells
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Cells were harvested at 4,000 rpm, and pellets were washed in ice-cold H2O and stored at −80°C. Pellets were resuspended in 11 mM Tris-HCl and 10 mM DTT and incubated on ice for 20 min. Cells were washed in ice-cold H2O, resuspended in spheroplasting buffer (1 M sorbitol, 50 mM Tris-HCL [pH 7.5], 1 mM CaCl2, 1 mM MgCl2, 14 mM β-mercaptoethanol, and 2,200 units Zymolyase T100), and incubated for 30 min at 37°C with shaking. Spheroplasts were sedimented in a fixed-angle rotor (Eppendorf F-34-6-38) at 6,000 rpm for 6 min, gently washed with 1 M sorbitol, transferred to 1.5 mL tubes, and sedimented for 1 min at 3,500 rcf at 4°C. Pellets were resuspended in 200 μL cold 0.4 M sorbitol, lysed on ice for 30 min by addition of 700 μL lysis buffer (25 mM HEPES/KOH [pH 8], 50 mM KCl, 10 mM MgSO4, 0.25% Triton X-100, 1 mM PMSF, 3 mM DTT, and protease inhibitor cocktail tablet [Roche]), and supplemented with 100 μg/mL RNaseA and 300 mM NaCl. Cell extracts were subsequently obtained by spinning the lysed spheroplasts at 12,000 rcf at 4°C for 5 min. Cleared lysates were loaded onto sucrose gradients prepared in a Biocomp gradient station and sedimented in a SW41 rotor (Beckman Optima L-100 XP Preparative Ultracentrifuge) at 18,000 rpm for 9 hr at 4°C. Gradients were fractionated into 30 equal fractions using a Biocomp gradient station.
7
Quantification and Characterization of EVs
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EVs were separated and quantified according to a previously published method [4] . This concentration method included a penultimate centrifugation step in Eppendorf polypropylene conical tubes (10,000 g for 30 min at 4 °C, in Eppendorf rotor F-34-6-38) that allowed the removal/isolation of lEVs. Subsequently, nano-sized sEVs, comprised mainly of exosomes, were pelleted in Beckman Coulter polypropylene open top tubes (118,000 g for 70 min at 4 °C, in Beckman rotor SW28). After washing, 10,000 or 118,000 g pellets were resuspended either in RIPA buffer or in PBS, for further immunoblotting or biophysics analyses, respectively. To estimate the amount of secreted vesicles, we quantified and compared the total protein content of the vesicle lysates using the BCA assay. Equal amounts of sEV lysates were loaded on SDS-PAGE in reducing (+2-Mercaptoethanol) and non-reducing conditions to evaluate the homodimer forms of CD9 [31] .
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