Kidneys were collected from C57BL/6 mice (3- to 5-week-old males). Cells were isolated from mice after perfusion with cold PBS plus EDTA and were cut into small pieces. Each kidney was digested in 6 ml RPMI1640 (GIBICO) with 1.5 mg/ml collagenase IV (Worthington) and 25 μl/ml DNase I (Sigma) for 30 min at 37°C with gentle shaking. After digestion, cells were filtered through a 100-µm strainer (BD). Cell suspensions were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 20 ng/ml Epidermal Growth Factor (Peprotech), 1× ITS (Insulin, Transferrin and Selenium, Gibco), and 1% penicillin-streptomycin (Corning) at 5% CO2 and 37 °C. When cell confluence reached 70%, the flowing cells were washed out and media were changed for the attached epithelial cells for another 48 hr.
100 m strainer
Manufactured by BD
Sourced in United States
The 100 µm strainer is a laboratory equipment designed to filter and separate particles or materials based on their size. It features a mesh or perforated screen with openings measuring 100 micrometers in diameter, allowing the passage of smaller particles while retaining larger ones. This strainer is commonly used in various laboratory applications to isolate, concentrate, or purify samples.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (2 mentions)
Transferrin, by Thermo Fisher Scientific (1 mentions)
Selenium, by Thermo Fisher Scientific (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Collagenase 4, by Worthington (1 mentions)
Dmem hg, by Thermo Fisher Scientific (1 mentions)
Deoxyribonuclease 1, by Merck Group (1 mentions)
Cellbind plate, by Corning (1 mentions)
Antibiotic antimitotic, by Thermo Fisher Scientific (1 mentions)
Percoll, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Cacl2, by Merck Group (1 mentions)
Pronase solution, by Merck Group (1 mentions)
Cytodex 1 microcarrier, by Merck Group (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
9 protocols using 100 m strainer
1
Isolation of Mouse Kidney Epithelial Cells
2
Villous Trophoblast Isolation from Placental Tissue
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Villous trophoblast cells were isolated from term placental tissue by enzymatic digestion and Percoll gradient separation, as previously described [13 (link)] with minor modifications. In brief, approximately 50 g of villous tissue was washed in 0.9% NaCl (Sigma, Saint Louis, MO, USA) four times for 5 min. Thereafter the tissue was minced and digested four times with 0.25% trypsin (Sigma, USA) and 300 IU/mL deoxyribonuclease I (Sigma, Saint Louis, MO, USA) at 37 °C (20 min each). The cell suspension was filtered and overlayed on fetal bovine serum (FBS, Seraglob, Schaffhausen, Switzerland). After centrifugation at 1000× g for 15 min at 10 °C, the cell pellet was collected in Dulbecco’s modified Eagle’s medium containing 4.5 g/L glucose (DMEM-HG, Gibco, Paisley, UK) basic medium (without FBS) and filtered through 100 µm strainer (BD Biosciences, Durham, NC, USA). Next, cells were overlayed on a discontinuous Percoll® (Sigma, Saint Louis, MO, USA) density gradient. After centrifugation, CTBs were located at the layer corresponding to 1.046–1.065 g/mL (35% to 50%) density [26 (link)]. The isolated cells were cultured at a density of 0.2 × 106 cells/cm2 and 0.5 × 106 cells/cm2 in 6-well or 24-well CellBIND plates (Costar, Kennebunk, ME, USA), respectively, using complete DMEM-HG medium (including 10% FBS and 1× antibiotic-antimitotic (Gibco, Grand Island, NY, USA).
3
Enzymatic Tumor Tissue Dissociation
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All experiments with human material were performed in accordance with the guidelines of the Declaration of Helsinki and were approved by the ethics committee of the Medical Faculty at the University Heidelberg (323/2004, Amendment 03). Informed consent was received from participants before study inclusion. Pieces of tumor tissue were collected from patients undergoing surgery at the Department of Surgery, University Hospital (Heidelberg, Germany) at 4 °C in PBS + 0.1 mg/mL penicillin/streptomycin (PBS/PS). Tumor tissue was minced into small pieces (1–2 mm in diameter), followed by three washings with 20 mL PBS/PS. Tumor pieces were incubated with 20 mL of digestion medium (1× medium 199 (Gibco, Darmstadt, Germany), 2 mg/mL collagenase IV (Invitrogen, Darmstadt, Germany) and 3mM CaCl2 (Sigma-Aldrich, München, Germany) at 37 °C for up to 150 min at constant rotation followed by filtering through a 100 µm strainer (BD Biosciences, Heidelberg, Germany). Leftovers on the strainer were further cultivated in vitro.
4
Chondrocyte Expansion on Cytodex® 1 Microcarriers
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Cytodex® 1 microcarriers (Sigma-Aldrich) were prepared according to the manufacturer’s protocol. Microcarriers were added to Corning® ProCulture® 125 ml glass spinner flask (Sigma-Aldrich) at a density of 10,000 microcarriers/ml in a total volume of 50 ml. TCP-passage 1 chondrocytes were seeded on microcarriers at an initial cell seeding density of 5000 cells/cm2. In the first 6 hours, microcarriers and chondrocytes were cultured under intermittent stirring at 25 rpm for 2 minutes for every 30 minutes. The culture was kept static in the next 18 hours before continuous stirring regime at 60 rpm. The dynamic microcarrier culture was maintained for 2 passages at 8 days each. Chondrocytes were harvested with 0.1% (w/v) pronase solution (Sigma-Aldrich) and separated from microcarriers by passing through a 100 µm strainer (BD Bioscience, USA). Cell preparation was observed under microscope to ensure all the microcarriers were removed.
5
Isolation and Expansion of Human Articular Chondrocytes
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Human articular chondrocytes (ACs) were isolated from knee joints of patients undergoing total knee replacement (MEC-2011-371 [surgical waste material], age: 58-68). Small chips were cut from macroscopically healthy areas of the cartilage using a scalpel and rinsed twice in PBS. The chips were then digested for 1 hour with 2 mg/ml protease (Sigma-Aldrich) in PBS followed by 0.5 mg/ml collagenase B (Roche Diagnostics, Rotkreuz, Switzerland) in DMEM (Lonza) with 10% heat-inactivated FCS (Thermo-Fisher) gentamicin [50 µg/mL] (Thermo-Fisher), and 1.5 µg/mL amphotericin B [Fungizone™] (Thermo-Fisher), rotating for at least twelve hours at 37°C.
Afterwards, the solution was filtered through a 100 µm strainer (BD Biosciences, Franklin Lakes, USA), centrifuged, and the pellet washed twice in PBS. Chondrocytes
were then plated at 7,500 cells/cm 2 , expanded in the above mentioned DMEM-based medium (refreshed twice a week) and passaged when 80% confluence was reached.
Chondrocytes were used for recellularization after one or two passages.
Afterwards, the solution was filtered through a 100 µm strainer (BD Biosciences, Franklin Lakes, USA), centrifuged, and the pellet washed twice in PBS. Chondrocytes
were then plated at 7,500 cells/cm 2 , expanded in the above mentioned DMEM-based medium (refreshed twice a week) and passaged when 80% confluence was reached.
Chondrocytes were used for recellularization after one or two passages.
6
Isolation of Bone Marrow Mononuclear Cells and Mesenchymal Stem Cells
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BM plugs were flushed gently from the marrow cavity of two femurs and two tibias using a syringe fitted with a 23-gauge needle containing cold phosphate-buffered saline (PBS) to isolate BM mononuclear cells. Flushed cells were then gently drawn up into and expelled from a 26-gauge needle to dissociate clumps. Next, cells were filtered through a 40-µm strainer (BD Falcon), and recovered total BM cells were counted. Intact BM plugs from two femurs and two tibias were enzymatically digested in prewarmed digestion solution (DNase I [100 µg/mL, Roche], LiberaseDL [250 µg/mL, Roche] in HBSS plus Ca2+and Mg2+) and incubated at 37 °C for 30 min with gentle shaking (~120 r.p.m.) to isolate MSCs. After weak vortexing for 20 s, cells were allowed to settle for ~3 min, after which the supernatant was transferred to another tube on ice. The settled BM plugs were enzymatically digested repeatedly two times, as previously described. The collected cells were then counted with a 100-µm strainer (BD Falcon).
7
Isolation and Characterization of ADMSCs
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Adipose-derived MSCs (ADMSCs) were obtained from adipose tissue from three healthy human donors as described previously (Ganbold et al., 2019 (link)). All experimental procedures in this study were approved by the Institutional Review Board of the School of Dentistry, Seoul National University (IRB No. S-D20150019). In brief, lipid tissue was collected from liposuction specimens, digested with 0.1% collagenase I (Gibco, Carlsbad, CA) in Hanks’ balanced salt solution (HyClone Laboratories, Logan, UT), and passed through a 100-µm strainer (BD Falcon, Franklin Lakes, NJ). Cells were resuspended in high-glucose Dulbecco’s modified Eagle’s medium (HyClone Laboratories) containing 10% fetal bovine serum (HyClone Laboratories). All cultures were maintained in a humidified incubator at 37°C and 5% CO2. Cells were passaged at ∼70% confluence, and cells at passages 3 to 7 were used. To generate MiBs, ADMSCs were seeded on AggreWell™400 plates (Stem Cell Technology, Cambridge, MA) and cultured for the 1, 2, 3, or 7 days to form MSC spheroids. To collect spheroids of uniform size, spheroids were passed through double-stacked cell strainers with pore sizes of 70 μm and 300 μm. MiBs were imaged using the JuLi stage system (NanoEnTek, Seoul, Korea), and MiB diameter was measured using ImageJ software (National Institutes of Health, version 1.53j, https://imagej.nih.gov/ij/download.html ).
8
Chicken Embryo Gonad Isolation and Analysis
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In ovo chicken embryos were incubated in a humidified egg incubator (Showafuranki) at 38°C, and automatic egg turning was performed. For ex ovo embryo culture, E2.5 embryos were excised from the yolk and cultured on a Petri dish following a method described by Uchikawa et al. (2004) (link) for observation. For embryo dissection, the embryo was isolated at E10, E12 or E16. Gonads were collected by gently picking them up with tweezers and then washing them once in cold PBS without Ca2+/Mg2+ (Takara Bio). Image and movie acquisition was conducted using a Leica M205 FA fluorescence histological microscope (Leica Microsystems) equipped with cellSens Standard (Olympus).
To prepare the sample for FACS analysis, gonad tissue was dispersed in TrypLE™ Express Enzyme (Gibco) with gentle shaking for 10 min. After centrifugation (600g , 5 min), dispersed tissue was resuspended in PBS without Ca2+/Mg2+ and passed through a 100 µm strainer (Falcon) for further analysis.
To prepare the sample for FACS analysis, gonad tissue was dispersed in TrypLE™ Express Enzyme (Gibco) with gentle shaking for 10 min. After centrifugation (600
9
Primary Hepatocyte Isolation via Collagenase Perfusion
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Primary hepatocyte isolation was performed using two-step collagenase perfusion technique, as previously described [27] . Briefly, a C57BL6/J mouse was anesthetized with isoflurane (Wako) and the inferior vena cava was exposed and cannulated with a 25-gauge needle. After clamping the superior vena cava and cutting the hepatic portal vein, the liver was perfused with 50 mL of Ca 2+ -free HBSS containing 0.5 mM EGTA (Wako) and 2 U/mL heparin (Novo-Heparin; Mochida, Tokyo, Japan) for 7 min, followed by 100 mL of collagenase solution containing 0.2% dispase II (Sanko Junyaku, Tokyo, Japan), 0.2% collagenase type II (Gibco, Palo Alto, CA, USA), 0.1 mg/mL DNase I (from bovine pancreas, Sigma), heparin 2 U/mL, 150 mmol/L NaCl, 5.4 mmol/L KCl, 0.34 mmol/L NaHPO 4 , 0.1 mmol/L MgSO 4 , 5.0 mmol/L CaCl 2 , 4.2 mmol/L, NaHCO 3 , 5.6 mmol/L glucose, and 10 mmol/L HEPES (all of the chemical reagents other than those indicated were purchased from Wako) for 7 min at 37°C. The liver was then resected out and gently shaken to retrieve the cells.
After filtering the isolated cells through a 100 µm strainer (BD Falcon, Franklin Lakes, NJ, USA), the cell suspension was washed three times by centrifugation at 50 × g for 3 min at 4°C. The isolated primary hepatocytes were immediately used for RNA extraction or the recellularization procedure.
After filtering the isolated cells through a 100 µm strainer (BD Falcon, Franklin Lakes, NJ, USA), the cell suspension was washed three times by centrifugation at 50 × g for 3 min at 4°C. The isolated primary hepatocytes were immediately used for RNA extraction or the recellularization procedure.
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