A total of 32 human biopsies were obtained from 32 donors (16 fetal and 16 adult). Fetal heart tissue was harvested under the supervision of the UCLA institutional review board, and adult human heart tissue was harvested from discarded pathological specimens at the time of heart transplantation under the supervision of the UCLA institutional review board or at the time of left ventricular assist device (LVAD) placement under the supervision of the UW institutional review board (Table S4 ). Biopsies were stored in Ca2+/Mg++-free Dulbecco’s PBS (DPBS; Cellgro) supplemented with 2% penicillin/streptomycin (P/S) and processed at a maximum of 4 hr postsurgery. Further details of heart dissociation and primary culture of HDCs are described in the Supplemental Experimental Procedures .
Dulbecco s pbs dpbs
Manufactured by Corning
Sourced in United States
Dulbecco's Phosphate Buffered Saline (DPBS) is a balanced salt solution used as a buffer in various cell culture and laboratory applications. It is designed to maintain the physiological pH and osmotic balance of cell cultures. DPBS is composed of inorganic salts, such as sodium chloride, potassium chloride, sodium phosphate, and potassium phosphate, formulated to provide a stable and isotonic environment for cells.
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Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Ack lysing buffer, by Lonza (1 mentions)
Astrocyte growth supplement, by ScienCell (1 mentions)
Primary human astrocytes, by ScienCell (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Corning (1 mentions)
Mycoalert plus kit, by Lonza (1 mentions)
Y27632, by Biorbyt (1 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Penicillin streptomycin p s, by Corning (1 mentions)
Acid citrate dextrose tubes, by BD (1 mentions)
Lymphocyte separation medium, by Corning (1 mentions)
9 protocols using dulbecco s pbs dpbs
1
Isolation of Human Heart Biopsies
2
Bone Marrow Engraftment in CD45.1 Mice
3
Isolation and Culture of Human Primary Astrocytes
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Human primary astrocytes were bought from ScienCell Research Laboratories (San Diego, CA, USA). Astrocytes were isolated from the cerebral cortex of a 22-week-old human donor. Cells were grown on poly-L-lysine-coated 75 cm2 culture flasks, in astrocyte medium supplemented with antibiotics (penicillin, streptomycin), astrocyte growth supplement and 2% FBS (all components from ScienCell Research Laboratories) in 5% CO2 and an increased-humidity atmosphere, at 37 °C. The culture medium was changed every three days, or daily for a culture confluency over 75%. When the culture reached 90% confluency, the cells were washed with Dulbecco’s PBS (DPBS, Corning, Corning, NY, USA), detached with 0.025% tripsin/EDTA (ethylenediaminetetraacetic acid) in DPBS and a 5%FBS/DPBS solution and centrifuged (150× g, 5 min, 20 °C).
4
Maintenance of hiPSC-Derived Cardiomyocytes
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Human-induced pluripotent stem cell-derived cardiomyocytes49 (link) were routinely maintained in E8 medium implemented with 10 μM Rho kinase inhibitor (Y27632; Biorbyt, Cambridge, UK) for the first 24 h after passage on 1:400 reduced growth factor Matrigel (Corning, Corning, USA). Cells were passaged ~1:15 every 3–4 days using 0.5 mM EDTA in Dulbecco’s PBS (DPBS; Corning, Corning, USA) after achieving ~80% confluence. Cell lines were used between passages 20 and 85. All cultures were routinely tested for mycoplasma using a MycoAlert PLUS Kit (Lonza, Basel, Switzerland).
5
Protein Binding Domain Screening in HEK293Ts
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HEK293Ts were maintained in DMEM with GlutaMax (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% (v/v) fetal bovine serum (GE Healthcare, Little Chalfont, UK) and 100 U/mL Pen Strep (Thermo Fisher Scientific). Dissociation of cells was achieved by washing with Dulbecco’s PBS (DPBS; Corning, Corning, NY) and incubation with Trypsin-EDTA 0.05% (v/v) (Thermo Fisher Scientific).
All transfections were performed with Lipofectamine LTX (Thermo Fisher Scientific) per manufacturer’s instructions at a 3:1 ratio of reagent to plasmid. For binding domain screening, 5 × 104 cells were plated in 24-well cluster plates. 1 μg of library clone plasmid was transfected either alone or with equimolar target plasmid along with an equal volume per weight of PLUS reagent. For protein extracts, 3 × 105 cells were plated in 6-well cluster plates and transfected with 2.5 μg plasmid with an equal volume per weight of PLUS reagent. At harvest, 10% of dissociated cells were separated for RNA and 90% for protein.
All transfections were performed with Lipofectamine LTX (Thermo Fisher Scientific) per manufacturer’s instructions at a 3:1 ratio of reagent to plasmid. For binding domain screening, 5 × 104 cells were plated in 24-well cluster plates. 1 μg of library clone plasmid was transfected either alone or with equimolar target plasmid along with an equal volume per weight of PLUS reagent. For protein extracts, 3 × 105 cells were plated in 6-well cluster plates and transfected with 2.5 μg plasmid with an equal volume per weight of PLUS reagent. At harvest, 10% of dissociated cells were separated for RNA and 90% for protein.
6
Isolated Lung Perfusion Protocol
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We purchased Ca2+- and Mg2+-containing Dulbecco’s PBS (DPBS) and Ca2+- and Mg2+-free PBS from Corning. Isolated lungs were perfused with HEPES-buffered vehicle of pH 7.4 and osmolarity 295 mOsm containing 150 mM Na+, 5 mM K+, 1 mM Ca2+, 1 mM Mg2+, and 10 mM glucose. Except where noted, fluorophores, reagents, and antibodies microinstilled in alveoli were dissolved or suspended in the same HEPES-buffered solution.
7
Fibroblast α-SMA Expression Analysis
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CRL-2097 fibroblasts were harvested with 0.05% trypsin and washed twice in Dulbecco’s PBS (DPBS; Corning Incorporated). Cells were fixed at 4°C for 20 min in cold methanol and washed in 1X DPBS. Blocking was performed for 30 min at room temperature in 5% bovine serum albumin (BSA; VWR International, Leicestershire, UK) in PBS with 0.05% Tween-20 (PBS-T) followed by a 30 min incubation in a primary antibody solution (1:200 dilution of α-SMA antibody) in PBS-T at room temperature. Cells were then washed again once in 1X DPBS and resuspended in a 1:500 dilution of secondary antibody solution in PBS-T (AlexaFluor488-conjugated goat anti-mouse IgG). Cells were counterstained with 500 ng/ml propidium iodide for 15 min at room temperature in the dark and analyzed on an Accuri C6 flow cytometer using Accuri-C6 analysis software (version 1.0.264.21; BD Biosciences, Franklin Lakes, NJ, USA). A total of 20,000 events were collected per sample.
8
Harvesting hMSM from Orthognathic Surgery
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hMSM samples were collected from three individuals (1 male and 2 females, 17-33 years of age) who underwent Le Fort I osteotomy as the orthognathic surgery between April and October 2016, with a discarded hMSM available. Informed consent was obtained, and all samples were collected in accordance with relevant guidelines under and ethically approved by the Ethics Committee at the Kyung Hee University Dental Hospital (approval no. KHD IRB 1509-1). Patients who neither had experienced nor were diagnosed with sinus pathology, maxillary neoplasm, metabolic diseases, genetic disease, nor had a history of previous sinus surgery were selected. After the collection, the samples were suspended in Dulbecco's PBS (DPBS; Corning, Inc.) containing 1% penicillin-streptomycin (PS; Corning, Inc.). Samples that were ~1x1 cm in size were used for cell culture.
9
PBMC Isolation from Venous Blood
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Peripheral blood mononuclear cells (PBMC) were obtained by venipuncture and collected into acid citrate dextrose tubes (Becton−Dickinson, Franklin Lakes, NJ) and further purified using Lymphocyte Separation Medium (Corning, Mediatech Inc., Manassas, VA). PBMC were washed 2–3 times in Dulbecco’s PBS (DPBS; Corning, Mediatech Inc.) to reduce platelet contamination.
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