All of the cell cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. BMSCs at passage 2 (P = 2) and ADSCs at P = 1 were purchased from Lonza. ASC52telo cells, hTERT-immortalized adipose-derived mesenchymal stem cells, were obtained from ATCC. HeLa cells were obtained from the Health Science Research Resources Bank (Osaka, Japan). BMSCs were cultured in an MSCGM BulletKit™, a mesenchymal stem cell basal medium supplemented with mesenchymal cell growth supplement, l -glutamine, and gentamycin/amphotericin-B (Lonza). ADSCs and ASC52telo cells were cultured in an ADSC-BulletKit™, an ADSC basal medium supplemented with the necessary supplements (Lonza). HeLa cells were cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS; Sigma), 0.1 mM non-essential amino acids (Gibco), 50 U/ml penicillin, and 50 μg/ml streptomycin (Gibco). Until they were used for cell growth analysis, cells were maintained in the medium as described above and passaged upon reaching 90% confluence.
Adscs
Manufactured by Lonza
Sourced in Switzerland
ADSCs are a type of laboratory equipment used for the isolation and cultivation of adipose-derived stem cells (ADSCs). These cells are obtained from the patient's own adipose tissue and can differentiate into various cell types, making them a valuable resource for regenerative medicine applications.
Automatically generated - may contain errors
Lab products found in correlation
Bmscs, by Lonza (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Six well plates, by Iwaki (1 mentions)
Collagen type 4, by Nitta Gelatin (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Gibco dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Mscgm bulletkit, by Lonza (1 mentions)
Gentamycin amphotericin b, by Lonza (1 mentions)
Hela cells, by Health Science Research Resources Bank (1 mentions)
Asc52telo cells, by American Type Culture Collection (1 mentions)
L glutamine, by Lonza (1 mentions)
Adsc bulletkit, by Lonza (1 mentions)
10 protocols using adscs
1
Characterization of Stem Cell Cultures
2
Isolation and Characterization of SHEDs
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Human SHEDs were isolated as described previously6 (link). Briefly, exfoliated deciduous teeth from 6- to 12-year-old individuals were extracted for clinical purposes and collected at Nagoya University School of Medicine. After separating the crown and root, the dental pulp was isolated and digested in a solution of collagenase type I (3 mg/ml) and dispase (4 mg/ml) for 1 h at 37 °C. Single-cell suspensions (1 × 104 to 2 × 104 cells/ml) were plated on culture dishes in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), then incubated at 37 °C in 5% CO2. SHEDs expressed a set of MSC markers (i.e., CD90, CD73, and CD105) but not endothelial and hematopoietic markers (i.e., CD34, CD45, CD11b/c, or HLA-DR). MSCs originated from human bone marrow (BMSCs) and human adipose tissue (ADSCs) were obtained from Lonza Ltd. SHEDs, BMSCs, and ADSCs were used at passage 8–10. At 70–80% confluence, these cells were washed with phosphate-buffered saline (PBS), the culture media were replaced with serum-free DMEM. After 48-h incubation, the media were collected and centrifuged for 3 min at 440 × g and 4 °C to remove detached cells, and the supernatants were then collected and centrifuged for 3 min, at 1,740 × g and 4 °C. These supernatants were collected and used for assays as SHED-CM, BMSC-CM, and ADSC-CM.
3
Co-culture of ADSCs and NHDFs for Keratinocyte Differentiation
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ADSCs (Lonza Group AG, Basel, Switzerland) were co-cultured with normal human dermal fibroblasts (NHDFs) as previously reported28 (link)30 (link). Briefly, NHDFs were seeded in six-well plates (IWAKI, Shizuoka, Japan) and cultured in Dulbecco's modified Eagle's Medium (DMEM) (Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) for 24 hours, while ADSCs were seeded on 0.4-µm Millicell® hanging cell culture inserts (Merck Millipore, Darmstadt, Germany) coated with type IV collagen (Nitta Gelatin, Osaka, Japan) and placed onto the plates. All-trans retinoic acid (Sigma-Aldrich) was added at 1 µM to the upper chamber. After culturing for 3 days, 25 ng/ml of bone morphogenetic protein 4 (R&D Systems, Minneapolis, MN, USA) was also added to the upper chamber. After 4 days, the media were replaced with keratinocyte serum-free medium (KSFM) (Thermo Fisher Scientific, Waltham, MA, USA). After 7 days of culture in KSFM, ADSCs were removed from the co-culture system and cultured on a dish coated with type IV collagen in KSFM for an additional 14 days.
4
Cytotoxicity of LF/TA-MSNs on ADSCs
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Prior to assessing the effect of LF/TA-MSNs on cellular activity, we conducted cytotoxicity tests on each group. For this, 1 × 104 adipose-derived stem cells (ADSCs, Lonza Group Ltd., Basel, Switzerland) were seeded in 96-well plates and cultivated in DMEM at 37 °C overnight. After that, cells were washed three times with PBS and exposed to one of the three sample of MSNs (100 μg/mL). At 1 and 3 days, DMEM was removed, and fresh PBS was added to wash the cells. Then, reagent from the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc. Rockville, MD, USA) was added to each well, and cells were cultured for 1 h in the dark at 37 °C. Absorbance at 450 nm was measured using a Multimode Reader. The control group consisted of untreated ADSCs. Cytotoxicity was evaluated as the percentage of viable cells versus the control group. The number of samples assessed per group was 4. All experiments for each time period were repeated three times.
5
Culturing Adipose-Derived Stem Cells
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ADSCs were purchased from Lonza (Australia) and cultured in cell CM consisting of Gibco Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher, Australia) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher, Australia), 1 ng/ml basic fibroblast growth factor (bFGF; Thermo Fisher, Australia), 1% penicillin-streptomycin (P/S; Thermo Fisher, Australia), 1% non-essential amino acids (NEAA; Thermo Fisher, Australia) in a cell culture incubator (37°C with humidified 5% CO2) as described previously (Li et al., 2017 (link)).
6
Adipose-Derived Stem Cell Seeding on Bone Cores
7
ADSC-Derived Conditioned Medium: Potentials
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Human adipose-derived stem cells (ADSCs) were purchased from Lonza (Basel, Switzerland). ADSCs were cultured in DMEM containing 20% FBS and penicillin/streptomycin. The cells that attached to the flask were then cultured, and the medium was changed every two days. Cells between the 3rd and 8th passages were used for all experiments. ADSCs were characterized as positive for CD73, CD90, and CD105 and negative for CD34 and CD45 by immunochemistry and flow cytometry. ADSCs have the potential to differentiate into adipocytes, osteoblasts and chondrocytes. To prepare CM from ADSCs, the cells were seeded at 5×105 cells per 10-cm plate. ADSCs reached 80% confluence and were then placed in serum-free medium for 24 h. The medium was then collected for in vitro and in vivo experiments. The collected CM was concentrated using Amicon Ultra-15 centrifugal filter units (Millipore, MA, USA). The final concentration of the CM was 4 μg/mL. In addition, ADSCs were transfected with miR-221/222 inhibitors for 24 h and the miR-221/222-inhibitors-ADSC-CM was collected to examine the effects of miR-221/222 on the apoptosis and fibrosis of H/R-treated cardiomyocytes.
8
Mesenchymal Stem Cells and Derived EVs
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Human bone marrow MSCs (BMSCs) and adipose MSCs (ADSCs) purchased from Lonza (Basel, Switzerland) were cultured in mesenchymal stem cell basal medium (MSCBM, Lonza, Basel, Switzerland) or ADSC growth medium (ADSCBM, Lonza, Basel, Switzerland), respectively. Cells were cultured for 15 days following thawing from passage one and then after every seven days for successive passages. Cells were used to isolate EVs until passage six for BMSCs and seven for ADSCs and viability after starvation was established around 90% for both cell types (90% ± 2% for BMSC and 92% ± 3% for ADSC). For in vitro experiments, dermal microvascular endothelium cells (HMEC-1) were obtained by the ATCC (Manassas, VA, United States) and cultures following manufacturer’s instruction with MCDB131 medium (Thermo Fisher Scientific, Waltham, MA, USA). Normal Human Dermal Fibroblasts (NHDF) are primary adult fibroblasts (Lonza, Basel, Switzerland). NHDF were cultured in FGM-2 Growth Media (Lonza, Basel, Switzerland) supplemented with bullet kit and 1 mL Mycozap PR (Lonza, Basel, Switzerland). Normal Human Epidermal Keratinocytes (adult skin) (NHEK) were purchased from Lonza and cultured with KGMTM Gold Keratinocyte Growth Medium (Lonza, Basel, Switzerland).
9
Ovarian Cancer Cell Line Cultivation and Spheroid Formation
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Ovarian cancer cell lines A2780 and A2780cis were from Sigma-Aldrich; SKOV3 (HTB-77) and COV318 were obtained from the American Type Culture Collection (ATCC); OVCAR5 (NIH) cells were provided by Dr. Baldassarre (CRO, Aviano, Italy); Kuramochi cells (JCRB0098), resistant to paclitaxel [15 ], were from JCRB Cell Bank; and OVCAR8 cells were from the National Cancer Institute Developmental Therapeutics Program Tumor Repository. All cell lines were routinely tested for mycoplasma, with negative results, and authenticated in our laboratory using PowerPlex 16 HS System (Promega, Madison, WI, USA) and GeneMapper ID version 3.2.1 to identify DNA short tandem repeats. ADSCs (Lonza) were maintained in Mesenchymal-Stem-Cell Growth Medium Bulletkit MSCGM (Lonza, Verviers, Belgium). Ovarian cancer cell lines were maintained in RPMI-1640 medium (Sigma-Aldrich Co., St. Louis, MO, USA) containing 10% heat-inactivated FBS, 100 U/mL penicillin and streptomycin (complete medium), at 37 °C in a 5% CO2 atmosphere. A2780cis cells were maintained in 1 µM cisplatin and cultured without the drug for 72 h before use in cellular assays. To obtain 3D-multicellular tumor spheroids (MCTSs), plates were coated twice with 20 mg/mL of poly(2-hydroxyethyl methacrylate) (poly-HEMA; Sigma) in 95% ethanol and washed once with PBS before cell seeding (2.0 × 104 cells in 24-well).
10
Culturing ADSCs and Breast Cancer Cell Lines
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ADSCs isolated from lipoaspirate of non-diabetic patients undergoing elective liposuction surgery were purchased from Lonza (Basel, Switzerland) and cultured in RPMI (Sigma-Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated foetal bovine serum (FBS) (Bovogen Biologicals, Melbourne, VIC, Australia) and 1% Penicillin-Streptomycin (PS) antibiotics (Hyclone Laboratories Inc, South Logan, Utah, USA). Breast tumour cell lines MCF-7 and MDA MB-231 were purchased from ATCC and cultured in the same medium as above. All cell types were cultured at 37 o C in the presence of 5% CO 2 . ADSCs were used within passages 2-8, whereas tumour cell lines were used at later passages >P10.
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