Human embryonic kidney (HEK) 293 cells (JCRB Cell Bank) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4 mM l -glutamine (Wako Pure Chemicals), 10% fetal bovine serum (FBS, One Shot™ fetal bovine serum, Thermo Fisher Scientific), and penicillin/streptomycin (PS) (Wako Pure Chemicals) at 37 °C, 5% CO2. Cells at 50–70% confluence in T25 flasks were treated with 0.25% trypsin-EDTA (Wako Pure Chemicals), harvested by centrifugation, and suspended in DMEM/10% FBS/PS. The cells were then washed with droplet EP medium before final re-suspension in the same medium and the cell concentration was adjusted. Four types of droplet EP medium were used: (1) DMEM with or without 10% FBS, (2) Dulbecco's phosphate-buffered saline without magnesium chloride and calcium chloride (d -PBS (-), Wako Pure Chemicals), (3) a low ionic strength 0.28 M mannitol, and (4) a mixture of d -PBS (-) and 0.28 M mannitol (at a 1:1 ratio).
D pbs
Manufactured by Fujifilm
Sourced in Japan, United States
D-PBS is a laboratory solution used for a variety of applications in scientific research and testing. It serves as a buffer that maintains the pH and osmolarity of cell culture media or other biological samples. D-PBS is a phosphate-buffered saline solution formulated to mimic the ionic composition of human body fluids.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Fujifilm (3 mentions)
L glutamine, by Fujifilm (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (3 mentions)
One shot fetal bovine serum, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin p s, by Fujifilm (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
D luciferin, by GoldBio (2 mentions)
Collagenase type 1, by Fujifilm (2 mentions)
Methanol, by Fujifilm (2 mentions)
Ammonium chloride solution, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium, by Fujifilm (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride edc, by Tokyo Chemical Industry (1 mentions)
Dylight 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Tyvek 1073b, by DuPont (1 mentions)
Alloderm, by Abbvie (1 mentions)
42 protocols using d pbs
1
HEK293 Cell Culture and Electroporation
2
Protein Extraction from CD45-Depleted Cells
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CD45-depleted cells were re-suspended in D-PBS(−) (Wako, Japan) and lysed by five freeze (in liquid N2)/thaw (in 37 °C water bath) cycles followed by centrifugation. Lysate was centrifuged at 15,000g for 30 min at 4 °C. The supernatant was recovered and the protein content was measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA).
3
Synthesis and Modification of PEG Polymers
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Alpha-methoxy-ω-amino PEG (MeO-PEG-NH2; Mn = 2400; Mw/Mn = 1.11; NOF, Tokyo, Japan) was purified using an ion-exchange CM Sephadex C-50 column (GE Healthcare, Buckinghamshire, UK) before use. Alpha-acetal-ω-amino PEG (acetal-PEG-NH2) was synthesized as described previously [27 (link)]. Beta-benzyl-l -aspartate N-carboxy-anhydride (BLA-NCA; NOF), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; Tokyo Chemical Industry, Tokyo, Japan), Cyclo[RGDfk(CX-)] (cRGD peptide, X = 6-aminocaproic acid, ∊-Acp; Peptide Institute, Osaka, Japan), sulfo-Cy3 mono-reactive dye (Lumiprobe, Orlando, Florida, USA), sulfo-Cy5 mono-reactive dye (Lumiprobe), DyLight488 NHS ester (Thermo Fischer Scientific, Waltham, Massachusetts, USA), methylamine solution (Me–NH2; 40%; Wako Pure Chemical Industries, Osaka, Japan), sodium hydroxide (NaOH; Koso Chemical, Tokyo, Japan), hydrochloric acid (HCl; Koso Chemical), acetic acid (CH3COOH; Nacalai Tesque, Tokyo, Japan), ferucarbotran (SPIO) solution (Resovist®, Fujifilm RI Pharma, Tokyo, Japan), Blocking one (Nacalai Tesque), 4% paraformaldehyde (4% PFA; Wako), Hoechst 33342 (Hoechst; Dojindo Laboratories, Kumamoto, Japan), and other reagents were used without further purification. PBS-Tween solution (0.1% v/v PBS-T) was prepared from Dulbecco’s phosphate-buffered saline (D-PBS; Wako) and Tween 20 solution (10% w/v, Wako).
4
Polyethylene Nonwoven Fabric for bFGF Adsorption
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A polyethylene nonwoven fabric (Tyvek® 1073B, DuPont, Wilmington, DE, USA), with a thickness of 180 µm, was used as a bFGF adsorbent. This fabric comprised of randomly oriented microfibers with a diameter of 0.5–10 µm (4 µm on average) and has a microporous structure with a Gurley-Hill porosity of 22 s/100 cc (range: 8–36 s/100 cc) according to the manufacturer’s product information sheet. As a bFGF source, 1 mg/mL human recombinant bFGF (bFGF AF, Katayama Chemical Industries Co., Ltd., Osaka, Japan) was used. For diluting the bFGF source and rinsing the bFGF-adsorbed membranes, phosphate-buffered saline (PBS) (D-PBS(–), FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was used.
5
Tilapia Scale Collagen Scaffolds for Oral Mucosa
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The EVPOMEs were manufactured by seeding oral mucosa keratinocytes obtained from five individuals onto microstructured tilapia scale collagen scaffolds having four microstructures with various aspect ratios (with one flat surface as a control), with or without 1% EDC crosslinking. AlloDerm (Allergan, Madison, NJ, USA) was used as a positive control15 (link). According to our human clinical application protocol, after presoaking the scaffolds in type IV collagen (5 μg/cm2, derived from the human placenta, Sigma-Aldrich) in D-PBS (Wako chemical, Osaka, Japan) overnight at 4 °C in a 12-well plate, oral mucosa keratinocytes were seeded onto all scaffolds at a cell density of 1.5 × 105 cells/cm255 (link). The composites were cultured in complete medium supplemented with 1.2 mM Ca2+ for 4 days in a submerged condition, and then raised to an air–liquid interface with the same culture medium for another 7 days15 (link).
6
Cigarette and Tobacco Smoke Extraction
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Smoke from one unit of C-cigarette or HTP was bubbled into 10 ml Dulbecco’s phosphate-buffered saline (D-PBS, Wako) at a speed of 10 s/50 ml pumping. CSE/HTPE solution was filtered (0.22 μm Milex-GS; SLGS033SB) to sterilize and remove insoluble particles. Commercial C-cigarettes and HTPs used are listed in Table 1 . Smoke from one C-cigarettes or HTP in 10 ml D-PBS was considered as 100% CSE/HTPE solution. 100% CSE or HTPE was diluted with an appropriate culture medium to make the indicated final concentration (v/v), depending on the experimental conditions. Condensed extract (200%) was generated by bubbling smoke from 2 units of HTPs directly into 10 ml culture medium, followed sterilization. Thereafter, 200% condensed extract was diluted with the same volume of culture medium to generate 100% HTPE.
7
Cell Line Mixing and Counting
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A human cell line, HEK293 (ATCC, CRL-1573), and mouse cell line NIH3T3 were cultivated in DMEM medium containing l -glutamine (Wako) supplemented with 10% FBS (Corning) and 1 × Antibiotic–Antimycotic (ThermoFisher Scientific). Cultivated cells were dissociated using 0.25% trypsin + EDTA (ThermoFisher Scientific). After washing by medium and D-PBS (–) (Wako), cells were suspended in PBS + 0.01% BSA (Sigma-Aldrich), and the cell suspension was filtered. Cell concentrations were measured using Countess IIFL (ThermoFisher Scientific). The same number of human and mouse cells were mixed, and the cell concentration was adjusted to 10 cells/µL with PBS-BSA.
8
Standardized E. coli Culture Protocol
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E. coli (ATCC25922) was used as a standard bacterial strain in these experiments. E. coli was picked from the glycerol stock (final concentration of glycerol, 15%) at −80 °C and cultured in 1% NaCl-LB broth (1% tryptone, 0.5% yeast extract) for up to 24 h at 37 °C with shaking (170 rpm) until reaching stationary phase after preculturing the day before. Bacterial culture medium was washed three times with d -phosphate buffered saline (D-PBS (−); Fujifilm Wako Chemicals) using a centrifuge at 12,000 rpm for 3 min each wash to remove the culture broth. Subsequently, bacterial suspension was diluted to an absorbance of 600 nm (A600) of 0.5 with D-PBS, and the adjusting bacterial solution after a 10-fold dilution (A600 = 0.5/10) was used for irradiation experiments.
9
Protein Extraction from Cultured Cells
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The cells were rinsed with D-PBS (-) (Fujifilm Wako) three times, harvested with TrypLE Express, and collected into 1.5-mL tubes. Next, 0.5 mL RPMI medium, GlutaMax Supplement, containing 2% (v/v) B-27 supplement, 1% (v/v) NEAA, and 1% (v/v) penicillin/streptomycin was added. The tubes were centrifuged at 3000 × g for 3 min, and the supernatant was removed. The cells were rinsed with cold D-PBS, followed by addition of 100 μL cold 1 × RIPA buffer (Cell Signaling Technology, Danvers, MA, United States) in double distilled water was added. The tubes were then vortexed, incubated on ice for 30 min, sonicated at 4°C (US-1R cleaner; AS ONE, Osaka, Japan), and centrifuged at 4°C, 10,000 × g for 20 min. The supernatant was collected into new 1.5-mL tubes and stored at −20°C.
10
Fabrication of Micropatterned Oral Mucosa Equivalents
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To manufacture the EVPOMEs, oral keratinocytes at p2 to p4 were seeded onto a micropatterned and a flat surface [as a control ex vivo produced oral mucosa equivalent (EVPOME)] collagen scaffold at a density of 1.5 × 105 cells/cm2. Briefly, the collagen scaffolds were presoaked with type IV collagen (Sigma-Aldrich, St. Louis, MO, USA) and D-PBS (Wako Chemical, Osaka, Japan) overnight at four °C in a 24 well-plate according to our human clinical application protocol [25 (link)]. After seeding oral keratinocytes onto the scaffolds, cell–scaffold composites were cultured in a submerged condition. They were fed daily with a complete medium supplemented with 1.2 mM Ca2+ for 4 days, raised to an air–liquid interface, and cultured with the same culture medium for another 7 days by refreshing the medium every other day. Nine pairs of EVPOMEs, formed on micropatterned and non-patterned control collagen scaffolds, were manufactured; during manufacturing, the identical EVPOME samples were examined using OCT imaging on days 8 and 11.
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