According to the policy approved by the local ethical committee, all tissue samples were obtained after informed consent. Human subcutaneous adipose tissue samples were obtained from lipoaspiration procedures. After washing, lipoaspirates were digested with 0.2% collagenase A type I solution (Sigma-Aldrich), under gentle agitation for 45 min at 37°C, and centrifuged at 800 ×g for 10 min to separate the Stromal Vascular Fraction (SVF) from adipocytes. If necessary, the SVF was treated with red blood cell lysis buffer for 5 min at 37°C and then centrifuged again. The supernatant was discarded and the cell pellet was resuspended and seeded in culture flasks in α-MEM supplemented with 10% heat-inactivated FBS, 100 units/mL penicillin, 100 units/mL streptomycin, and 2 mM L-glutamine and incubated at 37°C in a humidified atmosphere with 5% CO2. When the cultures were near confluence, the cells were detached by treatment with trypsin-EDTA 1X (Sigma-Aldrich), characterized, subcultured, and used at passage numbers 3–5.
Trypsin edta 1x
Manufactured by Merck Group
Sourced in United Kingdom, United States, France
Trypsin-EDTA 1X is a cell detachment solution used in cell culture applications. It is a mixture of the proteolytic enzyme trypsin and the chelating agent EDTA, which together disrupt cell-to-cell and cell-to-substrate adhesions, allowing cells to be harvested from a culture vessel.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Collagenase a type 1 solution, by Merck Group (1 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Sybrtm green master mix, by Thermo Fisher Scientific (1 mentions)
N cetyl n n n trimethylammonium bromide, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Cytoflex s cytometer, by Beckman Coulter (1 mentions)
Phalloidin, by Cytoskeleton (1 mentions)
Balanced salt solution bss, by Alcon (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Gentamycin, by GE Healthcare (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Attractene, by Qiagen (1 mentions)
Dntp mix, by Meridian Bioscience (1 mentions)
Purelink rna mini, by Thermo Fisher Scientific (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using trypsin edta 1x
1
Isolation and Characterization of Human Adipose-Derived Stromal Cells
2
Cell Culture and Molecular Analysis Protocol
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RPMI 1640 medium and Trypsin–EDTA (1X), antibiotics (penicillin and streptomycin), and DAPI staining solution (4,6-diamidino-2-phenylindole) were purchased from Sigma-Aldrich Co. (Poole, UK). Fetal bovine serum (FBS) was obtained from Gibco Life technology, (Paisley, UK). TRIzol® reagent and SYBRTM Green master mix were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Primers were designed by oligo software version 7 and synthesized via Bioneer Company (Daejeon, Korea). The solutions for RNA extraction and cDNA synthesis were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Annexin V-FITC apoptosis detection kit was purchased from EMD Chemicals (Gibbstown, USA).
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RPMI 1640 medium and Trypsin–EDTA (1X), antibiotics (penicillin and streptomycin), and DAPI staining solution (4,6-diamidino-2-phenylindole) were purchased from Sigma-Aldrich Co. (Poole, UK). Fetal bovine serum (FBS) was obtained from Gibco Life technology, (Paisley, UK). TRIzol® reagent and SYBRTM Green master mix were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Primers were designed by oligo software version 7 and synthesized via Bioneer Company (Daejeon, Korea). The solutions for RNA extraction and cDNA synthesis were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Annexin V-FITC apoptosis detection kit was purchased from EMD Chemicals (Gibbstown, USA).
3
NIH-3T3 Cell Line Apoptosis Assay
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The NIH-3T3 cell line was obtained from the national cell bank of Iran (Tehran, Iran). Cell culture flasks and plates were purchased from JET BioFIL (Elgin IL, USA). Cell culture dishes were obtained from TPP (Trasadingen, Switzerland). RPMI1640 medium and Trypsin – EDTA (1X) were obtained from Sigma-Aldrich (Poole, UK). Normal melting agarose and fetal bovine serum (FBS) were bought from Invitrogen-Gibco (Paisley, UK). The other chemical materials such as paraformaldehyde, H2O2, 2% lysis buffer (Tris-HCl 100 mM, EDTA 20 mM, NaCl 1.4M, C-TAB (N-Cetyl-N,N,N-trimethylammonium bromide) were bought from Merck (Kenilworth, NJ, USA). Annexin V-FITC apoptosis detection kit was obtained from EMD Chemicals (Gibbstown, NJ, USA).
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The NIH-3T3 cell line was obtained from the national cell bank of Iran (Tehran, Iran). Cell culture flasks and plates were purchased from JET BioFIL (Elgin IL, USA). Cell culture dishes were obtained from TPP (Trasadingen, Switzerland). RPMI1640 medium and Trypsin – EDTA (1X) were obtained from Sigma-Aldrich (Poole, UK). Normal melting agarose and fetal bovine serum (FBS) were bought from Invitrogen-Gibco (Paisley, UK). The other chemical materials such as paraformaldehyde, H2O2, 2% lysis buffer (Tris-HCl 100 mM, EDTA 20 mM, NaCl 1.4M, C-TAB (N-Cetyl-N,N,N-trimethylammonium bromide) were bought from Merck (Kenilworth, NJ, USA). Annexin V-FITC apoptosis detection kit was obtained from EMD Chemicals (Gibbstown, NJ, USA).
4
Quantifying Liposome Uptake in Pancreatic Tumor Cells
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The internalization of rhodamine-labeled liposomes by mouse pancreatic tumor cells was determined by flow cytometry. Cells were seeded at a density of 5 × 104 cells/well on 96-well plates (COSTAR®, Corning Inc., New York, NY, USA) and rhodamine-labeled-liposomes at 100 µM were added and incubated at different time points (30 min, 1, 3 and 5 h). Then, cells were washed with PBS and detached with Trypsin-EDTA 1X (Sigma-Aldrich, San Luis, Missouri, USA). The action of trypsin was neutralized with complete culture medium and cells centrifuged at 1200 rpm for 5 min and washed with PBS twice. Finally, cells were resuspended in 200 μL of PBS and analyzed on the Cytoflex S cytometer (Beckman Coulter, CA, USA) using the FL3 channel. The final data was represented as median fluorescence intensity (MFI).
5
In Vitro Endothelial Cell Characterization
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Immortalized CEC line B4G12 (ACC 647) was purchased from DSMZ (Braunschweig, Germany). Human Endothelial Serum-Free Medium (SFM), 24 well tissue culture plates, ZO-1 antibodies and AlexaFluor 488 Goat anti-mouse IgG (H+L) antibodies were purchased from Fisher Bioblock Scientific (Vaulx-Milieu, France). Fibroblast Growth Factor (FGF-2), calcein, sodium dodecyl sulfate (SDS), propidium iodide (PI), trypsin/EDTA 1X and Hoechst 33342 were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Balanced Salt Solution (BSS) was purchased from Alcon (Rueil-Malmaison, France). Black carbon nanoparticles of 15-nanometer diameter (Black Pearls 470) were purchased from Cabot Corp and Business (Billerica, MA). Ti:Sa laser system was purchased from Thalès (Neuilly-sur-Seine, France). ANT-130 XYZ motorized stage and A3200 Software-Based Machine Controller were purchased from Aerotech (Pittsburgh, PA). The inverted fluorescence microscope was purchased from Olympus (Tokyo, Japan). FACSCalibur flow cytometer was purchased from Becton Dickinson (Franklin Lakes, NJ). Gentamycin was purchased from PAA Laboratories, (Pasching, Austria). Phalloidin was purchased from Cytoskeleton (Denver, CO).
6
Transient Transfection of HEK293T Cells
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Human embryonic kidney (HEK293T) cells were transiently transfected in triplicate with 1.2 µg of OPN1LW-pMT4 recombinant expression vector using Attractene (Qiagen, Chadstone, VIC, Australia) in six-well cell culture plates. After 48 h, transfected cells were harvested using Trypsin-EDTA 1X (Sigma-Aldrich, Castle Hill, NSW, Australia), and washed four times with PBS (1X; 138 mM NaCl, 2.70 mM KCl, 10 mM NaPO4, 1.8 mM KPO4, pH 7.4). Total RNA was extracted using the PureLink RNA Mini with the TRIzol kit (Thermo Fisher Scientific, Scoresby, VIC, Australia), before the generation of oligo dT-primed cDNA using 5 µl (1–2 µg) of total RNA incubated with 5 µl oligo dT (500 ng) and 20.5 µl sterile RNase-free water for 15 min at 85 °C, followed by 2 min on ice. Subsequently, 10 µl of 5X First-Strand Buffer (Genesearch, Arundel, QLD, Australia), 5 µl (0.1 M) of DTT (Genesearch, Arundel, QLD, Australia), 2.5 µl (10 mM) dNTP mix (Bioline, Alexandria, NSW, Australia), and 1 µl RNase murine inhibitor (40 U/µl; Genesearch, Arundel, QLD, Australia) was added and incubated for 2 min at 25 °C. Then, 1 µl of M-MuLV Reverse Transcriptase (Genesearch, Arundel, QLD, Australia) was added and incubated for a further 5 min at 25 °C, followed by 1 h at 42 °C. Finally, a further 1 µl of M-MuLV Reverse Transcriptase (Genesearch, Arundel, QLD, Australia) was added and incubated for another 1 h at 42 °C.
7
Passaging and Expansion of Adipose-Derived Stem Cells
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Upon reaching 70% confluence in the T175 flask (Thermo Fisher Scientific, Waltham, MA, USA), the ASCs were washed with phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) and trypsinized using 3 mL 0.5% trypsin-EDTA 1x (Sigma, St. Louis, MO, USA). Trypsin was neutralized using 7 mL DMEM/F12 medium, 10% FBS medium and 50 µg/mL Gentamicin (Invitrogen, Carlsbad, CA, USA), and removed by centrifugation at 300 RCF for 5 min. The cells were again seeded at a density of 5000–7000 cells/cm2 in DMEM/F12 medium plus 10% FBS, and maintained at 37 °C with 5% CO2. ASCs were passaged by seeding at 5000–7000 cells/cm2, the medium was changed every third day and the cells were grown to 70% confluence before splitting. ASCs cultivated to passage 3–4 were used in this study.
8
Quantification of Caspase Activation in PC3 and A375 Cells
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PC3 and A375 cells were grown for 48 hrs in presence or absence of 50 μg/mL fraction II. Cells were enzymatically detached using Trypsin-EDTA 1x (Sigma) solution and centrifuged at 1000 rpm for 5 min.
Pelleted cells were lysed with 70 μL of 1% Triton X-100 solution in PBS, incubated 10 min at room temperature, and successively centrifuged at 10,000 rpm for 10 min. The amount of protein extract in the supernatant medium was quantified using Bradford microassay method (BioRad, Segrate, Milan, Italy).
PC3 and A375 cells extracts corresponding to 20 μg protein extracts were used to detect the presence of activated caspases 3/7/8, using Ac-Asp Glu-Val-Asp-MCA peptide (Pepta Nova, 380 Peptide Institute). The degree of caspase activation was quantified by spectrofluorimetric readings using Spectra Max Gemini EM-500 (Molecular Devices) and elaborated by SoftMax Pro 5.2 software.
Untreated PC3 and A375 cells were used as negative control while cells treated with Doxorubicin (DXR, Ebewe Pharma) at 5 μM for 24 h were used as positive control.
Pelleted cells were lysed with 70 μL of 1% Triton X-100 solution in PBS, incubated 10 min at room temperature, and successively centrifuged at 10,000 rpm for 10 min. The amount of protein extract in the supernatant medium was quantified using Bradford microassay method (BioRad, Segrate, Milan, Italy).
PC3 and A375 cells extracts corresponding to 20 μg protein extracts were used to detect the presence of activated caspases 3/7/8, using Ac-Asp Glu-Val-Asp-MCA peptide (Pepta Nova, 380 Peptide Institute). The degree of caspase activation was quantified by spectrofluorimetric readings using Spectra Max Gemini EM-500 (Molecular Devices) and elaborated by SoftMax Pro 5.2 software.
Untreated PC3 and A375 cells were used as negative control while cells treated with Doxorubicin (DXR, Ebewe Pharma) at 5 μM for 24 h were used as positive control.
9
RPMI1640 Cell Culture and mRNA Extraction
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RPMI1640 medium and Trypsin –EDTA (1X) were obtained from Sigma-Aldrich (Poole, UK). Fetal bovine serum (FBS) and Trizol reagent were acquired from Invitrogen-Gibco (Paisley, UK). RNase inhibitor, dNTP, reverse transcriptase and SYBR green PCR Master Mix were obtained from Fermentas (St. Leon-Rot, Germany), and Applied Biosystems (Warrington, UK), respectively. Tris-HCl and DEPC water were purchased from Sinagen co. (Tehran, Iran). Red blood cells lysing buffer (RLB) was prepared by 2M Tris-HCl pH 7.6, 1M MgCl2, 3M NaCl; and 10 mM Tris-HCI (pH 8.0). Other materials were purchased from Merck (Darmstadt, Germany). All solutions including 10% (w/v) PEG 4000/6000, LiCl and 3 M potassium acetate (pH 5.2) and TE buffer were prepared using DEPC water.
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RPMI1640 medium and Trypsin –EDTA (1X) were obtained from Sigma-Aldrich (Poole, UK). Fetal bovine serum (FBS) and Trizol reagent were acquired from Invitrogen-Gibco (Paisley, UK). RNase inhibitor, dNTP, reverse transcriptase and SYBR green PCR Master Mix were obtained from Fermentas (St. Leon-Rot, Germany), and Applied Biosystems (Warrington, UK), respectively. Tris-HCl and DEPC water were purchased from Sinagen co. (Tehran, Iran). Red blood cells lysing buffer (RLB) was prepared by 2M Tris-HCl pH 7.6, 1M MgCl2, 3M NaCl; and 10 mM Tris-HCI (pH 8.0). Other materials were purchased from Merck (Darmstadt, Germany). All solutions including 10% (w/v) PEG 4000/6000, LiCl and 3 M potassium acetate (pH 5.2) and TE buffer were prepared using DEPC water.
10
Osteogenic Differentiation of hBMSCs on Peptide-Grafted Surfaces
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Commercially available hBMSCs (Lonza, France) were grown on gelatin coated culture flasks in MSCs growth medium (MSCGM) (Lonza, France), subcultures using trypsin/EDTA 1x (Sigmaaldrich, France) and maintained in a humidified atmosphere containing 5 % CO2 at 37 °C. Modified glass substrate of 1x1 cm² were sterilized with 70 % ethanol and rinsed by PBS, then placed in cell culture plates 24 well. To induce osteogenic differentiation, hBMSCs at passage 4 were seeded on different peptides grafted surfaces at a density of 104 cells/cm2 for 6 h in serum free α-MEM medium (Life technology, France). This allows the interactions between grafted peptides and their cell surface receptors without hassle of serum proteins. Serum-free medium was then removed and replaced with α-MEM medium containing 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin. The culture media was changed twice per week during four weeks of cell culture.
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