An amount of 7 g of gelatin (Sigma‐Aldrich) was dissolved by heating in 70 mL of Dulbecco's Phosphate‐Buffered Saline (DPBS) at 50°C. Afterwards, 7 mL of methacrylic anhydride (Sigma‐Aldrich) was added dropwise. After 3 h of reaction, 200 mL of DPBS (Gibco Laboratories) was added to dilute the mixture, and the whole solution was stirred for another 15 min at 50°C. Subsequently, the solution was dialyzed against deionized water in a dialysis tube (Shanghai Rebus Network Technology Co., Ltd.) with a cut‐off molecular weight of 12–14 kDa for 1 week at 37°C. The deionized water was replaced every 2 days. Lastly, solid GelMA was obtained by lyophilization of the dialyzed solution and subsequent storage at room temperature.
Dulbecco s phosphate buffered saline dpbs
Manufactured by Merck Group
Sourced in United States, Germany, Italy, United Kingdom, Belgium, France, Denmark, Brazil
Dulbecco's phosphate-buffered saline (DPBS) is a balanced salt solution commonly used in various laboratory applications. It is a sterile, isotonic buffer solution that maintains the pH and osmolarity of cell cultures and other biological samples. DPBS is designed to preserve the viability and physiological functions of cells during experimental procedures.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (28 mentions)
Methacrylic anhydride, by Merck Group (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (9 mentions)
L glutamine, by Merck Group (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Fetal bovine serum (fbs), by Merck Group (9 mentions)
Penicillin, by Merck Group (8 mentions)
Streptomycin, by Merck Group (8 mentions)
Trypsin edta, by Merck Group (8 mentions)
Triton x 100, by Merck Group (8 mentions)
Trypsin edta, by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Merck Group (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Trypan blue, by Merck Group (6 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (6 mentions)
Fetal bovine serum (fbs), by PAN Biotech (6 mentions)
Trypsin, by Thermo Fisher Scientific (6 mentions)
258 protocols using dulbecco s phosphate buffered saline dpbs
1
Synthesis of Photocrosslinkable Gelatin Methacrylate
2
Osteogenic Differentiation Marker Assay
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The early-stage marker of osteogenic differentiation was measured using an ALP assay (Abcam, Cambridge, MA). Similar to the proliferation assay, MC3T3-E1 preosteoblasts were seeded on PLCL, PLCL/Col, PLCL/MXene, and PLCL/Col/MXene nanofibrous matrices at a density of 1.0 × 104 cells/matrix and then incubated in basal media for up to 14 days. ALP activity was determined by measuring the transformation of ρ-nitrophenyl-phosphate (ρNPP) to ρ-nitrophenol (ρNP) in yellow color, which is produced in the presence of ALP [39 (link)]. At the end of pre-determined incubation period, the cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS, Sigma-Aldrich Co.) and incubated in a 0.1% Triton X-100 solution (Sigma-Aldrich Co.) in Tris-buffer (10 mM, pH 7.5, Sigma-Aldrich Co.) for 10 min. Subsequently, a freshly prepared ρNPP solution (50 μl) was added to cell lysate (80 μl) of each matrix, followed by incubation for 1 h in a CO2 incubator. After incubation, the reaction was finished by adding a stop solution (20 μl). The absorbance was measured at 405 nm using a microplate reader and the ALP activity was calculated from the ρNP formation (μmol) divided by the volume (ml) and reaction time (min).
3
Serum Survival Assay for ST258
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To test survival of ST258 clinical isolates in normal human serum (NHS) ± antibiotic pretreatment, bacteria were cultured in LB to an OD600 of ~0.2 and treated with antibiotics (Table S2) for 2 h. Antibiotic-free LB cultures were used as controls. After antibiotic pretreatment, bacteria were washed with Dulbecco’s phosphate-buffered saline (DPBS) (Sigma-Aldrich, St. Louis, MO), and 40 μL of washed bacteria was mixed with 360 μL of NHS to yield ~107 bacteria in 90% NHS. Cultures were incubated at 37°C for 30 min with continuous shaking (1,200 rpm). Culture aliquots were serially diluted and plated on LB agar plates. Colonies were enumerated the following day and used to determine CFU per milliliter. The percentage of survival ± antibiotic at 30 min was determined relative to CFU per milliliter at 0 min (start of the assay) by using the following equation: % survival = CFU/mL30 min/CFU/mL0 min × 100.
4
Cell Culture Protocol for Three Cancer Lines
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Three cell lines, A549 (human lung cancer), LLCMK2 (monkey kidney cells) and HepG2 (human liver cancer), were cultured in Dulbecco's Modified Eagle Medium (DMEM; 1×; Sigma Aldrich, Dublin, Ireland) supplemented with 10% MSC‐Qualified FBS (Sigma Aldrich, Dublin, Ireland) and 1% penicillin–streptomycin (Penstrep; GIBCO, ThermoFisher, Dublin, Ireland) at 37 °C in a 5% CO2 incubator. The A549 and HepG2 cell lines were obtained from ATCC (Manassas, VA, USA). The LLMCK2 cell line was a generous donation from U. Power, Queen's University Belfast, Northern Ireland. The cells were sub‐cultured every 3–4 days, when their confluency reached 60–80%. For subculturing, briefly, the growth medium was removed, and cells were washed with 10 mL of Dulbecco's Phosphate‐Buffered Saline (D‐PBS, containing no calcium, magnesium, or phenol red; Sigma Aldrich) to remove any remnant medium and cell secretions. Then, cells were Trypsinised (Trypsin; GIBCO, ThermoFisher), seeded at the appropriate volume (1–1.5%) and further incubated at 37 °C in a 5% CO2.
5
HeLa Cell Culture Preparation
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HeLa cells used in the experiments (93021013 Sigma-Aldrich, St. Louis, MO, USA) were kept in Dulbecco’s Modified Eagle’s Medium (DMEM), with 10% fetal bovine serum (Biowest SAS, Nuaillé, France), 4 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin in an incubator with a humidified atmosphere containing 5% CO2 at 37 °C. Before micropipette experiments, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS, Sigma-Aldrich, St. Louis, MO, USA ) and detached by applying 0.05% (w/v) trypsin and 0.02% (w/v) EDTA solution for 2 min in the incubator. Cells were resuspended in DMEM, and the concentration was measured using a Bürker counting chamber. HeLa cells with a density of 104 cells per mL were pipetted in surface-coated Petri dishes. Subsequently, the Petri dishes containing the cells were incubated at 37 °C with 5% CO2 for 90 min to allow for adhesion.
6
Biopolymer-Based Biomaterial Fabrication
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List of the chemicals used in this study are as follows: Gelatin type A from porcine skin (G2500, Sigma Life Sciences, St. Louis, MO, USA), sodium alginate (VIVAPHARM® alginate PH163 S2, from brown algae, with approval as a pharmaceutical excipient, JRS PHARMA GmbH & Co. KG, Rosenberg, Germany), methacrylic anhydride (MA, Sigma-Aldrich, St. Louis, MO, USA), Dulbecco’s Phosphate Buffered Saline (DPBS, Sigma-Life Science), poly (d, l-lactide-co-glycolide) (PLGA 75:25, 66,000–107,000, Sigma-Aldrich), polyvinyl alcohol, (PVA, 86–89% low molecular weight ThermoFisher, Kandel, Germany), calcium chloride dihydrate (CaCl2·2H2O, Carl Roth, Karlsruhe, Germany), dichloromethane (DCM, >99.8%, Sigma-Aldrich), sodium periodate (Carl Roth), ethanol absolute (VWR International), ethylene glycol (Carl Roth), and lithium phenyl-2,4,6-trimethyl-benzoyl phosphinate (LAP, Tokyo Chemical Industry (TCI), Tokyo, Japan).
7
Cell Culture Medium Composition
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The cell culture medium was Dulbecco’s modified eagle medium with 4.5 g/L glucose (DMEM, Merk Life Science, Milano, Italy), 10% decomplemented fetal bovine serum (FBS, Life Technologies, Monza, Italy), and 1% penicillin/streptomycin (P/S, Life Technologies, Monza, Italy). Dulbecco’s phosphate-buffered saline (DPBS) and trypsin-EDTA were purchased from Sigma. Dimethyl sulfoxide (DMSO) was purchased from Life Technologies. Cell culture reaction tubes and cell culture plates and flasks were purchased from Corning Inc. (Corning, NY, USA). The freezing containers used for cryopreservation were Nalgene purchased from Merk Life Science.
8
Isolation and Culture of Murine Bone Marrow-Derived Macrophages
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Harvest of murine bone marrows was performed as previous described, using 2–3 months-old male C57BL/6 mice (JAX) (Zhang et al., 2008 (link); Qadri et al., 2018 (link)). Harvest of murine bone marrows was approved by the IACUC committee at Chapman University. Cells were centrifuged at 400xg, 4°C for 10 min. The supernatant was discarded, and cells were resuspended and seeded in T75 flasks at ∼4.0 × 106/mL in 10 ml pre-warmed complete media and incubated at 37°C in 5% CO2 incubator. On days 3 and 5, the supernatant was discarded, and cells were washed with 4 ml Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma Aldrich). On day 7, the supernatant was discarded, and BMDMs were incubated with cell stripper nonenzymatic cell dissociation solution (Sigma Aldrich) at 37°C for 5 min and cells were gently scraped. An equal volume of complete media was added, and stripped cells were centrifuged at 400xg, 4°C for 10 min. The supernatant was discarded, and macrophages were resuspended in complete media.
9
Apoptosis Induction in Cancer Cells
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Apo-B-α-LA was procured from Sigma Aldrich Chem. Co., and used in all the experiments without further purification. The lanthanum perchlorate salt was prepared starting from lanthanum oxides followed by treating the reaction mixture with perchloric acid and recrystallizing the product. A 10 mM Tris-HCl buffer at pH 7.4 was used for all the experiments unless otherwise mentioned.
Both the healthy and the cancer cells used in the present study were obtained from National Centre for Cell Science, Pune, India. Dulbeco’s Modified Eagle Medium (DMEM) and DMEM without phenol red, Dulbecco’s Phosphate Buffered Saline (DPBS), Fetal Bovine Serum (FBS) and coverslips for fluorescence microscopy were purchased from Sigma-Aldrich, USA. Caspase 3/7 green detection reagent was procured from Thermo Fischer scientific.
Both the healthy and the cancer cells used in the present study were obtained from National Centre for Cell Science, Pune, India. Dulbeco’s Modified Eagle Medium (DMEM) and DMEM without phenol red, Dulbecco’s Phosphate Buffered Saline (DPBS), Fetal Bovine Serum (FBS) and coverslips for fluorescence microscopy were purchased from Sigma-Aldrich, USA. Caspase 3/7 green detection reagent was procured from Thermo Fischer scientific.
10
Subcutaneous Injection of Tumor Cells
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To prepare injection aliquots, cells were detached from the flasks with trypsin. Once the cells were visibly detached, DMEM was added to inactivate the Trypsin. Cells were then pooled in a 50 mL Falcon tube, and an aliquot was removed to perform a cell count. The Falcon tube of cells was centrifuged for 5 min at 4 °C. Following centrifugation, the supernatant was removed, and the cells were resuspended in Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma, Oakville, ON, Canada) to the proper concentration. The cell solution was aliquoted into designated microcentrifuge tubes for each group and diluted 1:1 with Corning® Matrigel Matrix (Millipore Sigma, Oakville, ON, Canada) on ice to reach the final injection concentration. On day 6, each mouse received a 50 µL subcutaneous injection into the right flank which delivered 1 × 106 EO771 cells.
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