A primary normal human dermal fibroblast (HDFn) cells (ATCC PCS-201-010) were kindly provided by Dr. Toshiya Nakamura (Hirosaki University, Aomori, Japan) and an immortalized mouse embryonic fibroblast (m5S) cells (JCRB1322) were kindly provided by Dr. Seiji Kodama (Osaka prefecture University, Osaka, Japan). The HDFn cells were cultured in Dulbeccos’s modified eagle medium (DMEM; Invitrogen) supplemented with 10% exosome-depleted foetal bovine serum (Exo-FBS, System Biosciences), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The m5S cells were cultured in alpha-MEM supplemented with 10% Exo-FBS (System Biosciences), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cell cycle phase was measured by Muse Cell Analyzer, using a Muse Cell Cycle Assay Kit (Merck Millipore) following the manufacturer’s instructions.
Exo fbs
Manufactured by System Biosciences
Sourced in United States, Italy
Exo-FBS is a fetal bovine serum (FBS) product that is depleted of extracellular vesicles (EVs). It is designed to be used as a culture supplement in cell culture applications where the presence of EVs from the FBS may interfere with experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Type 1 collagen, by BD (3 mentions)
Ebm 2 medium, by Cambrex (3 mentions)
Exoquick tc, by System Biosciences (2 mentions)
Dmem f12, by Merck Group (2 mentions)
Exoquick tc exosome isolation reagent, by System Biosciences (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Total exosome isolation reagent, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Muse cell cycle assay kit, by Merck Group (1 mentions)
Penicillin, by System Biosciences (1 mentions)
Streptomycin, by System Biosciences (1 mentions)
Hexadecyltrimethylammonium bromide ctab, by Merck Group (1 mentions)
Silver nitrate agno3, by Merck Group (1 mentions)
Exosome precipitation kit, by System Biosciences (1 mentions)
Poly allylamine hydrochloride pah, by Merck Group (1 mentions)
Sodium borohydride nabh4, by Merck Group (1 mentions)
Bioultra, by Merck Group (1 mentions)
L ascorbic acid bioxtra, by Merck Group (1 mentions)
Gold 3 chloride trihydrate haucl4 3h2o, by Merck Group (1 mentions)
34 protocols using exo fbs
1
Fibroblast Cell Cycle Analysis
2
Gold Nanoparticle Synthesis and Characterization
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Gold(iii ) chloride trihydrate (HAuCl4·3H2O, ≥99.9%, Sigma-Aldrich), sodium borohydride (NaBH4, >98%, Sigma-Aldrich), hexadecyltrimethylammonium bromide (CTAB, ≥99%, Sigma-Aldrich), sodium citrate (tribasic dehydrate, C6H5Na3O7·2H2O, ≥99%, Sigma-Aldrich), poly(allylamine hydrochloride) (PAH, M.W. 17,500 g mol−1, Sigma-Aldrich), poly(acrylic acid) sodium salt (PAA, M.W. 15,000 g mol−1, Sigma-Aldrich), silver nitrate (AgNO3, 99.0%, Sigma-Aldrich), l -ascorbic acid (BioXtra, ≥99.0%, crystalline, Sigma-Aldrich), hydrochloric acid (HCl, certified 1.0 N, Fisher Chemical), phosphate-Buffer Saline (PBS, 1×, Corning), exosome-depleted FBS (Exo-FBS, SBI), trypan blue solution (0.4%, Invitrogen), Triton X-100 solution (BioUltra, Sigma-Aldrich), exosome precipitation kit (Exo-quick, SBI). All solutions were prepared with nanopure water.
3
Culturing Human Dermal Fibroblasts
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Human dermal fibroblasts (HDF) cells (Sigma Aldrich) were cultured in T-75 flasks containing Dulbecco's modified eagle medium (DMEM, without phenol red), nonessential amino acids and 1% penicillin-streptomycin solution (Mediatech), along with 10% exosome-depleted FBS (Exo-FBS, SBI) in a humidified incubator with 5% CO2 at 37 °C.
4
Differentiation of THP-1 Monocytes to Macrophages
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THP-1 human monocytes (American Type Culture Collection, ATCC/TIB-202) were cultured at 37 °C at 5% CO2 in complete RPMI (cRPMI) media. This media contained RPMI 1640 base medium (ATCC) supplemented with 2-mercaptoethanol (Gibco) (final concentration of 0.05 mM) and 10% exosome-depleted fetal bovine serum (EXO-FBS, SBI). Monocytes (2.5 × 105 cells/ml) were activated to macrophages (MΦ) using 200 nM of Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 72 h. After the activation, unbound cells and remaining media were removed, and the adherent cells were washed three times using phosphate buffer saline (PBS).
5
Exosome Isolation and Purification
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Cells at 50–60% confluence were detached with 2 mm EDTA/PBS, washed 2 times with PBS and then cultured in DMEM supplemented with 5% Exo‐FBS (SBI, Palo Alto, CA, USA) until reaching 80–90% confluence. The cell culture supernatants were collected and centrifuged at 300 g for 10 min, 2000 g for 10 min, 10 000 g for 30 min and 100 000 g for 70 min. Exosomes were washed with PBS at 100 000 g and centrifuged for 70 min, and the pellet containing exosomes preserved.
6
Extracellular Vesicle Isolation from Serum-free Media
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EVs were isolated from the culture media of cells grown in serum–free medium supplemented with 10% Exo-FBS (FBS depleted of EVs, SBI, System Biosciences, CA, USA) in 150 mm plates (15 mL medium volume) with Cell Culture Media Extracellular Vesicle Purification Kits (Norgen, Biotek Corp, Ontario, Canada), according to the manufacturer’s instructions.
7
Extracellular Vesicle Isolation from Cell Lines
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HeLa, 293T, and U-2 OS (ATCC HTB-96) cells were maintained at 37°C, 5% CO2, in DMEM media supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 1% Pen/Strep (100 U/mL penicillin, 100 μg/mL streptomycin), and 1mM sodium pyruvate. For EV collection, cells were seeded at 1–2 x 104 cells/cm2 in DMEM media supplemented with 10% exosome-depleted FBS (Exo-FBS, SBI), L-GlutaMAX, 1% penicillin/streptomycin, and sodium pyruvate. After 3 days, conditioned culture media was collected for EV isolation and corresponding cells were collected and resuspended in PBS for RNA isolation.
8
Isolation of Exosomes from MDA-MB-231 Cell Media
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Exosomes were isolated from cell culture media of MDA-MB-231 cells. 4 × 106 cells were plated in 150 × 25 mm cell culture dishes (Corning, 430599) with their usual growth medium (described in the previous section) to allow plate attachment. After 24 h, cells were washed twice with PBS (Dulbecco’s PBS, Sigma-Aldrich, Cod. D8537, lot RNBH3372), and 12 mL of RPMI medium (Sigma-Aldrich) supplemented with 10% FBS depleted of exosomes (Exo-FBS, SBI, Cod. Exo-FBS-250A-1, lot 161004-002), 1% A/A, and 1% amphotericin B was added. After 48 h, culture media were collected and centrifuged at 3,000 × g for 15 min at room temperature (RT) to remove cellular debris. The supernatants were transferred into new sterile tubes, and the appropriate volume of the ExoQuick-TC exosome isolation reagent (SBI, Cod. EXOTC50-A) was added according to the manufacturer’s instructions. Then the tubes were gently mixed until the separation between the two phases was no longer visible. The tubes were kept standing at 4°C overnight (O/N). The following day, the tubes were centrifuged first at 1,500 × g for 30 min and then at 1,500 × g for 5 min to ensure complete removal of the ExoQuick-TC solution. Last, exosome pellets were resuspended in 300 μL of PBS solution.
9
Exosome Isolation from Cell Cultures
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For bulk cells, cultures were grown until reach a 60% confluence. Then, cells were grown during 48 hours in DMEM-F12 supplemented with 5% exosome-depleted FBS (Exo-FBS, SBI). Later, culture medium was recovered and centrifuged at 2000 x g for 5 min, to eliminate cells, apoptotic bodies and debris, and then at 16500 x g for 20 min at 4°C to eliminate microvesicles [62 (link)]. For prostatospheres cultures, serum free culture medium was recovered after 21 days of growth and centrifuged at 2000 x g for 5 min and later the supernatant at 16500 x g for 20 min at 4°C. Later, exosomes were isolated by precipitation with the reagent ExoQuick-TC (SBI), according to manufacturer's instructions. Briefly, 10 ml of culture medium was mixed with 2 ml of ExoQuick-TC and incubated overnight at 4°C. Later, the mix was centrifuged at 1500 x g for 30 min at room temperature to obtain an exosome pellet.
10
Exosome-Induced Small RNA Sequencing
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NFs (5 × 105) were plated in 100-mm dishes with DMEM-F12 culture medium supplemented with 10% Exo-FBS (SBI) and 1% A/A and treated with 120 μg of MDA-MB-231-derived exosomes and the same volume of PBS solution as a control. After 24 h, cells were collected, and RNA was extracted using TRIzol reagent (Life Technologies, 15596018). Samples were shipped to Genomix4Life (Baronissi, Salerno, Italy), who performed small RNA sequencing using Illumina HiSeq2500 (SmallRNA 1 × 20M Cod. G4L1630 – iMir, Cod. G4L15055) and bioinformatics analysis (PCA component and differential expression analysis). Two biological replicates for each experimental point were analyzed. For the statistical analysis, p < 0.05 alone was considered for experimental significance; no p value adjustment was performed because of the small sample size (NFs from pt. #1).
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