Isolated EVs solutions were diluted 1:100 in Dulbecco’s PBS (DPBS; Life Technologies, Grand Island, NY, USA), and 5 μL of diluted exosomes were placed on parafilm before the Formvar/Carbon-coated grid are placed on top of exosome drops and allowed to stand for 2 min. Grids with adherent exosomes were washed three times with 30 μL DPBS drops and fixed with 2% paraformaldehyde in DPBS for 5 min. Finally, grids were incubated with 30 μL drops of 2% uranyl acetate and examined by electron microscopy [54 (link)]. The samples were washed with distilled water seven times (2 min each), and then they were viewed under a FEI Tecnai F30 Twin (Atlanta, GA, USA) transmission EM to measure the size of the isolated EVs [54 (link)].
Dulbecco s pbs dpbs
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Dulbecco's Phosphate Buffered Saline (DPBS) is a commonly used buffer solution in cell culture applications. It is a balanced salt solution that maintains the osmotic balance and pH of the cellular environment. DPBS is formulated to provide the essential ions and nutrients required for the survival and growth of cells in vitro.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Veritas microplate luminometer, by Promega (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Rankl, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
90 protocols using dulbecco s pbs dpbs
1
Isolation and Characterization of Extracellular Vesicles
2
Quantifying Intestinal and Blood Microbiome
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Samples of peripheral blood were cultivated log 10 and diluted by PBS. Jejunum (40 cm of the proximal part of the jejunum) and ileum (40 cm segment of a terminal part of the small intestine containing the ileum and part of the distal jejunum) lavages were cut off, filled with 2 mL of Dulbecco’s PBS (DPBS; Life Technologies, Carlsbad, CA), gently kneaded, and rinsed. Colon lavage was obtained by placing the whole colon in a 90 mm Petri dish, cut into small pieces in 4 mL of DPBS. All lavages were vigorously vortexed. Further, 0.2 g of mesenteric lymph nodes, liver, and spleen were homogenized in 0.8 mL deionized water in a 2 mL Eppendorf tube containing two 3.2 mm stainless-steel beads in a TissueLyser LT beadbeater (Qiagen, Hilden, Germany) shaken for 3 min at 50 Hz. The intestinal lavages, tissue homogenates, and blood were serially diluted in PBS and cultivated in 90 mm Petri dishes with MRS agar for lactobacilli (Oxoid), MacConkey agar (Merck, Darmstadt, Germany) for E. coli or Brilliant green agar (Oxoid) for S. Typhimurium. The plates were incubated aerobically at 37 °C for 48 h for lactobacilli or 24 h for E. coli or S. Typhimurium. The CFU were counted from dishes optimally containing 20–200 colonies.
3
RNA Extraction and qPCR Analysis
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The cells were lysed directly in the wells by addition of 300 μl of Buffer RLT supplemented with 15 mM β-mercaptoethanol (Thermo Fisher Scientific) after a wash with Dulbecco’s PBS (DPBS, Life Technologies). Total RNA was isolated using the RNeasy Mini extraction kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s protocol. RNA levels and quality were quantified using a NanoDrop spectrophotometer. cDNA synthesis was performed by Thermo Fisher Scientific Verso cDNA Synthesis Kit and Quantitative PCR assay was performed by Sybergreen Gene Expression Assays in triplicate wells of 96-well plates. Primers are listed in table S8 and 2−(Cp GOI−Cp internal gene) was used for analysis. The housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase) was selected to control for variation in cDNA amounts.
4
Single Cell Suspension Preparation
5
Reconstitution and Validation of Immune Agonists
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R848 (TLR7/8 agonist; InvivoGen) was reconstituted in sterile water at a concentration of 3 mM. TDB (Mincle agonist; Avanti Polar Lipids) was reconstituted in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at a concentration of 20 mg/mL, heated at 60 °C for 30 s, then brought to 2 mg/mL using sterile Dulbecco’s PBS (DPBS) without Ca2+, Mg2+, and phenol red (Life Technologies) and heated again at 60 °C for an additional 15 min. 3 M052 (TLR7/8 agonist) was a kind gift from Dr. Mark Tomai, 3M Drug Delivery Systems.
PKC-δinhibitor peptide (SFNSYELGSL; Anaspec Inc.), PKC inhibitor (Gö 6983; Abcam) and Clathrin inhibitor (Pitstop®1, and corresponding negative control; Abcam) was reconstituted in DMSO at 10 mM and used at 10 μM concentration in cell culture.
All agonists and inhibitors were verified to be endotoxin-free (<1 EU/ml), as measured by Limulusamebocyte lysate (LAL) assay per the manufacturer’s instructions (Charles River). Sterile DPBS or DMSO were included in experiments at appropriate concentrations as a negative control when indicated.
PKC-δinhibitor peptide (SFNSYELGSL; Anaspec Inc.), PKC inhibitor (Gö 6983; Abcam) and Clathrin inhibitor (Pitstop®1, and corresponding negative control; Abcam) was reconstituted in DMSO at 10 mM and used at 10 μM concentration in cell culture.
All agonists and inhibitors were verified to be endotoxin-free (<1 EU/ml), as measured by Limulusamebocyte lysate (LAL) assay per the manufacturer’s instructions (Charles River). Sterile DPBS or DMSO were included in experiments at appropriate concentrations as a negative control when indicated.
6
High-Affinity Dopamine Transporter Assay
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We measured H3DA uptake to examine the function of the high affinity DA transporter (DAT) using standard techniques [43 (link)]. Specifically, cultures from the SN and VTA were treated on DIV4. Cultures were incubated for 1 hr in media at 37°C containing a vehicle of sodium citrate/PBS, 40 μM nomifensine, an inhibitor of DAT, or the combination of CDNF and N4 (100 mg/ml each). After the 1 hr incubation, the solution was changed to Dulbecco’s PBS (DPBS) (Life Technologies) containing 5 μM glucose and 100 nM H3DA at 37°C for 15 min. The cultures were then placed on ice and washed 3 times with cold DPBS with glucose followed by addition of an extraction solution consisting of 33% ethanol and 0.4 N perchloric acid for 15 min at 37°C to release the total tritium. The extraction solution was then collected into scintillation vials filled with ScintiSafe cocktail (Thermo Fisher Scientific). Total tritium present in the solution was then assessed on a scintillation counter (LS6500, Beckman Coulter, Pasadena, CA, USA).
7
Immunostaining of H. pylori-infected cells
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One day prior to experiments cells were seeded at 5 x 104 cells in a 24-well plate equipped with uncoated cover slides and grown overnight at 37°C and 5% CO2. Cells were infected with Hp wild type or isogenic mutant strains with an MOI of 10 for 3h at 37°C and 5% CO2. For immunostaining cells were fixed with 4% PFA for 10 min at room temperature. Cells were washed twice with Dulbecco´s PBS (DPBS, Life Technologies) and blocked overnight with 2% FCS in PBS at 4°C. Fixed cells were incubated with mouse anti-CEACAM5 (26/3/13, Genovac, 1:300), rabbit anti-Hp (AK175, 1:400) and rat anti-integrin beta1 (AIIB2, Millipore, 1:200) for 1h at room temperature. After washing secondary antibodies were applied (goat anti-rat Alexa488, goat anti-mouse Alexa555 and goat anti-rabbit Alexa647 all from Invitrogen, 1:1000) and incubated for 1h at room temperature in the dark. Cell nuclei were stained with DAPI (5μg/ml) for 10 min. Samples were mounted on the cover slip with Fluorescent Mounting Medium (DAKO). A cytospin3 (Shandon) was used to centrifuge suspension cells onto glass slides. Micrographs were taken with a confocal laser scanning microscope (LSM880, Zeiss) with Airyscan Module using a 63x oil immersion objective.
8
G-CSF and Busulfan Effects on Mouse Spermatogenesis
9
Isolation of MSCs from WBS Patients
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To isolate MSCs from a patient with WBS (WBS_MSCs), we rinsed the thymus tissue of a WBS patient with Dulbecco’s PBS (DPBS; Life Technologies, Darmstadt, Germany) and cut it into small pieces. The tissue pieces were then incubated with 5 mL of 0.1% collagenase II solution (Merck Millipore, Darmstadt, Germany) in DMEM low glucose (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C. The solution containing tissue pieces was briefly mixed every 30–60 min. After 3 hr, the tissue suspension was filtered through a 100-μm tissue strainer and the filtrate was centrifuged for 5 min at 300 × g. After centrifugation, the supernatant was aspirated and the cell pellet was resuspended in 30 mL of MSC medium and seeded in three T175 cell culture flasks. The cells were then incubated in 25 mL medium at 37°C and 5% CO2. The obtained cells are called WBS_MSCs.
10
Extracellular Vesicle Isolation Protocol
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EVs were isolated from the supernatants using the Total Exosome Isolation kit (Life Technologies, USA) per manufacturer’s guidelines. All experiments were performed with EVs at a 1:100 dilution. Cell supernatants of the different cell types were centrifuged at 3000× g and 4 °C for 30 min. Pellets were discarded and for each 1 mL of supernatant, 0.5 mL of precipitation buffer was added. The mix was vortexed and incubated overnight at 4 °C. The solution was then centrifuged at 10,000× g and 4°C for 1 h and 15 min and pellets were resuspended in fresh filtered (0.22 µm) Dulbecco’s PBS (DPBS; Life Technologies, Grand Island, NY, USA). EVs stocks were stored at −20 °C.
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