Liver NPCs and KCs were isolated from normal or IR livers of B6 mice by in situ collagenase perfusion. In brief, livers were perfused via the portal vein with calcium- and magnesium-free HBSS supplemented with 2% heat-inactivated FBS, followed by 0.27% collagenase IV (Sigma). Perfused livers were dissected and teased through 70 μm nylon-mesh cell strainers (BD Biosciences). NPCs were separated from hepatocytes by centrifuging at 50g for 2 minutes 3 times. NPCs were stained with fluorescence-labeled Abs and analyzed by FACS. To enrich KCs, NPCs were suspended in HBSS and layered onto a 2-layer 25%–50% Percoll gradient (Sigma-Aldrich) in a 50 mL conical centrifuge tube and centrifuged at 1800g at 4°C for 15 minutes. KCs in the middle layer were collected and allowed to attach to cell culture plates in supplemented DMEM with 10% FBS for 15 minutes at 37°C. Nonadherent cells were removed by replacing the culture medium. The purity of KCs in the adherent cells was determined by immunofluorescence staining with anti–F4/80. Most (80%–90%) adherent cells were F4/80 positive.
Nylon mesh cell strainer
Manufactured by BD
Sourced in United States
The Nylon mesh cell strainer is a laboratory equipment used for filtration and separation of cells and other particulates from a liquid suspension. It features a nylon mesh screen that allows the liquid to pass through while retaining the desired solids.
Automatically generated - may contain errors
Lab products found in correlation
Rbc lysis buffer, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Collagenase 4, by Merck Group (2 mentions)
Collagenase, by Fujifilm (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Novaseq 6000 system, by Illumina (2 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Percoll gradient, by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor bfgf, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Ter119 apc, by BD (1 mentions)
Cd71 fitc, by BD (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
28 protocols using nylon mesh cell strainer
1
Isolation of Liver NPCs and KCs
2
Isolation of Primary Hepatic Macrophages and Bone Marrow-Derived Macrophages
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Primary hepatic macrophages were isolated as described.29 (link) In brief, mouse livers were perfused in situ with 37°C warmed HBSS solution, followed by a collagenase buffer (collagenase type IV, Sigma-Aldrich). Perfused livers were dissected and teased through 70 μm nylon mesh cell strainers (BD Biosciences). The nonparenchymal cells were separated from hepatocytes and layered onto a 50%/25% 2-step Percoll gradient (Sigma-Aldrich). After centrifugation, hepatic macrophages in the middle layer were collected and allowed to adhere to cell culture plates in DMEM with 10% FBS, 10 mM HEPES, 2 mM GlutaMax, 100 U/mL penicillin, and 100 μg/mL streptomycin for 15 min at 37°C. Murine primary BMDMs were isolated from mice.29 (link) In brief, bone marrow cells were harvested from the femurs and tibias of Notch1FL/FL and Notch1M-KO mice and cultured in DMEM supplemented with 10% FBS and 20% L929-conditioned medium.
3
Spleen Cell Isolation and Whole Blood Collection
4
Isolation of Liver Non-Parenchymal Cells
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Liver NPCs were isolated from normal or IR livers of B6 mice by in situ collagenase perfusion. In brief, livers were perfused through the portal vein with calcium‐ and magnesium‐free Hank's balanced salt solution (HBSS) supplemented with 2% heat‐inactivated fetal bovine serum (FBS), followed by 0.27% collagenase IV (Sigma‐Aldrich). Perfused livers were dissected and filtered through 70‐μm nylon mesh cell strainers (BD Biosciences, San Diego, CA). NPCs were separated from hepatocytes by low‐speed centrifugation (50g, 2 minutes) 3 times. NPCs were stained with fluorescence‐labeled antibodies and analyzed by FACS. To enrich KCs, NPCs were suspended in HBSS and layered onto a two‐layer 25% to 50% Percoll gradient (Sigma‐Aldrich) in a 50‐mL conical centrifuge tube and centrifuged at 1,800g at 4°C for 15 minutes. KCs in the middle layer were collected and allowed to attach to cell‐culture plates in supplemented Dulbecco's modified Eagle's medium (DMEM) with 10% FBS for 15 minutes at 37°C. Nonadherent cells were removed by replacing the culture medium. The purity of KCs in the adherent cells was approximately 80%, as determined by immunofluorescent staining with anti‐F4/80.
5
Isolation of Immune Cell Populations
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Cell suspensions of spleen, thymus and LN were prepared by collagenase D digestion and passage through nylon mesh cell strainers (BD Biosciences). BM was collected from tibias and femurs. Whole blood was isolated by cardiac puncture for serum and flow cytometry. Red blood cells were lysed with RBC lysis buffer (Sigma-Aldrich). Intraepithelial cells from small intestine were isolated as described with modifications50 . Briefly, small intestines were sectioned after removal of Peyer’s patches and incubated for 20 min at room temperature in HBSS, 10% bovine calf serum, EDTA and HEPES with mild agitation (two rounds). Supernatants were passed through nylon mesh strainers and centrifuged over a 44%-67% Percoll gradient.
6
Isolation of Immune Cell Populations
7
Isolation and Characterization of Colonic and Splenic Cells
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Colonic tissues were treated with HBSS containing 1 mM dithiothreitol, 20 mM ethylenediaminetetraacetic acid (EDTA) and 12.5 mM HEPES at 37 °C for 30 min on a stirrer. The tissues were then minced and dissociated in RPMI-1640 containing 0.5 mg ml−1 collagenase (Wako), 0.125 mg ml−1 DNase I, 2% new-born calf serum, 100 U ml−1 penicillin, 100 µg ml−1 streptomycin and 20 mM HEPES (2 R media) at 37 °C for 30 min to obtain single-cell suspensions. Single splenic leucocyte suspensions were prepared by mechanically disrupting the tissues through 100-µm nylon-mesh cell strainers (BD Biosciences) in 2 R media. These preparations were then subjected to flow cytometry analysis using the LSR II flow cytometer (BD Biosciences).
8
Isolation and Preparation of Murine Thymocytes
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Freshly excised thymi from mice were gently teased with a syringe and forceps in DMEM containing 10 mM HEPES (pH 7.2–7.5). The mechanically disrupted cell clumps were poured through 70 μm nylon mesh cell strainers (BD Falcon, Bedford, Massachusetts) to remove connective tissue and prepare single cell suspensions. The cell suspensions were washed once with PBS, and the red blood cells were lysed with Ammonium-Chloride-Potassium (ACK) buffer. The remaining thymocytes were counted, washed twice in PBS, and recovered in DMEM containing 10% fetal calf serum, 2 μM glutamine, 100 U/ml each of penicillin and streptomycin, and 10 mM HEPES (pH 7.2–7.5) for 1 h, followed by further treatments as indicated.
9
Isolation and Characterization of Mesenchymal Stem Cells
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Fresh specimen samples were minced with a scalpel in minimal essential medium-α (MEMα; Mediatech, Herndon, VA, USA) and passed through a series of 100-µm nylon mesh cell strainers (BD Falcon, Franklin Lakes, NJ, USA). The resulting cell suspensions were combined and washed twice in serum-free MEMα, and then cultured in complete MSC medium consisting of MEMα, 10% fetal bovine serum (Gibco, Invitrogen, Carlsbad, CA, USA), 2-mM L-glutamine (Mediatech), and 1X antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA, USA). After 24 h, nonadherent cells were removed by washing twice with phosphate-buffered saline (Mediatech) and cultured until they reached confluence. Thereafter, cells were trypsinized (0.25% trypsin with 0.1% ethylenediaminetetraacetic acid) and sub-cultured at a density of 5000 cells/cm2. The obtained isolated cells were assessed for several mesenchymal features, including adhesion to plastic, tri-lineage differentiation, and the presence of typical MSC surface markers (positive for CD105, CD90, and CD72; negative for CD45, CD31, and NG2).
10
Isolation and Culture of Glioma Cells
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Fresh tumor specimens were obtained in the operating room from glioma patients undergoing surgery. Each specimen was place in a sterile centrifuge tube (SPL Life Sciences Co., Ltd, Korea) on ice, and was weighed on the same electronic precision balance (Sartorius® TE4101-L, Sartorius Weighing Technology GmbH, Goettingen, Germany) within 1 h. Thereafter, specimens were processed using a previously reported mechanical dissociation method [1 (link), 3 (link), 17 (link)]. Briefly, surgical specimens were minced and dissociated with a scalpel in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12; Mediatech, Manassas, VA, USA) and then passed through a series of 100-μm nylon mesh cell strainers (BD Falcon, Franklin Lakes, NJ, USA). Cell suspensions were washed twice in DMEM/F-12 and cultured in complete media (DMEM/F-12) containing 1xB27 supplements (Invitrogen, San Diego, CA, USA), 20 ng/ml of basic fibroblast growth factor (bFGF; Sigma, St. Louis, MO, USA), 20 ng/ml of epidermal growth factor (EGF; Sigma), and 50 U/ml penicillin/50 mg/ml streptomycin [1 (link), 3 (link), 17 (link)].
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