The NP tissues were washed three times with phosphate-buffered saline (PBS; Gibco, Grand Island, NY, United States), minced into small fragments and digested in 0.25% (w/v) trypsin (Gibco) and 0.2% (w/v) type Π collagenase (Gibco) and then placed in PBS for approximately 3 h at 37°C in a gyratory shaker. Cells were filtered through a 70 μm mesh filter (BD, Franklin Lakes, NJ, United States). Primary NP cells were cultured with growth medium (Dulbecco’s Modified Eagle’s Medium) and Ham’s F-12 Nutrient Mixture (DMEM-F12; Gibco), 20% (v/v) fetal bovine serum (FBS; Gibco), 50 U/mL penicillin, and 50 μg/mL streptomycin (Gibco) in 100 mm culture dishes in a 5% (v/v) CO2 incubator. The cells were passaged at approximately 80% (v/v) confluence using trypsin and subcultured in a 60 mm culture dish (2.5 × 105 cells/well). Cells that had been passaged no more than twice were used in the subsequent experiments.
70 μm mesh filter
Manufactured by BD
Sourced in United States
The 70 μm mesh filter is a laboratory equipment designed for filtration purposes. It features a mesh size of 70 micrometers, which is suitable for various filtration applications that require this specific pore size.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Corning (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Nacalai Tesque (1 mentions)
Penicillin streptomycin, by Nacalai Tesque (1 mentions)
Liberase, by Roche (1 mentions)
Rpmi 1640 media, by GE Healthcare (1 mentions)
Collagenase 2, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
7 aad, by BioLegend (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Facsaria 2, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Lsrii running diva software, by BD (1 mentions)
6 protocols using 70 μm mesh filter
1
Primary Intervertebral Disc Cell Isolation
2
Preparation of Inflammatory Synovial Cells
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Inflammatory synovial cells were prepared by cutting the synovial tissues from arthritic joints into small pieces, followed by enzymatic digestion for 30 min at 37°C in plain IMDM buffer (Sigma-Aldrich) with Liberase TM (0.25 mg/ml; Roche), and then by mashing the digested tissues through 70-μm mesh filter (BD Biosciences). The resultant single cell suspension of synovial tissues was used for flow cytometry or cell sorting experiments. Non-hematopoietic synovial cells adherent to culture dishes were passaged several times without any stimulation in DMEM medium (Nacalai Tesque) supplemented with 20% FBS (GIBCO) and penicillin-streptomycin (Nacalai Tesque) in order to remove synovial immune cells and used as FLS.
3
Gingival Tissue Isolation for Flow Cytometry
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Gingival tissue was harvested during periodontal surgery from an inflamed site with a persistent periodontal pocket ≥6 mm in periodontitis patients (n = 6). In the controls, gingival tissue was harvested from a site without periodontal pocket in conjunction with an implant surgery or tooth extraction performed for reasons other than periodontitis (n = 6). Tissues were placed in complete RPMI 1640 media (GE Healthcare Life Sciences, Uppsala, Sweden). The tissues were cut into small pieces and incubated with 0.3 mM CaCl2 in phosphate buffered saline (PBS) at 37°C with magnetic stirring for 10 min. After that, they were incubated with Collagenase II (0.5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and DNase I (0.25 mg/ml; Roche, Basel, Switzerland) in RPMI 1640 without FCS at 37°C with magnetic stirring for 40 min. The suspension was filtered through a 70-μm mesh filter (Falcon, Corning, NY, USA), washed with complete RPMI, and centrifuged at 400 g for 5 min. Cell suspensions were then stained and analyzed by flow cytometry.
4
Isolation of CD4+CD8+ T Cells
5
Cortical Neuron Culture Preparation
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Cortical neuron cultures were prepared from mouse embryos (E18). The cerebral cortex was isolated and maintained in cold Hank's Balanced Salt Solution (HBSS, Gibco) supplemented with 0.45 % glucose. After removal of the meninges, the cortical tissue was digested mildly with trypsin for 17 min at 37°C and mechanically dissociated. Cells were washed three times in HBSS and resuspended in Neurobasal medium supplemented with 2 mM Glutamax (Gibco) before filtering in 70 μm mesh filters (BD Falcon). Cells were plated onto glass coverslips (5 × 104 cells/cm2) coated with 0.1 mg/ml poly-L-lysine (Sigma). 2 h after seeding, the plating medium was replaced by complete growth medium (Neurobasal medium supplemented with 2% B27 (Invitrogen) and 2 mM Glutamax) and the coverslips were incubated at 37°C in a humidified 5 % CO2 atmosphere. Every 3–4 days, half of the conditioned medium was removed and replaced by fresh growth medium. Primary cultures were transfected with Lipofectamine 2000 on day 7 in vitro (7 DIV), according to the manufacturer's, instructions and the cells were fixed 72 h after transfection. All the experimental procedures were carried out according to European Union guidelines (Directive 2010/63/EU) and following protocols that were approved by the Ethics Committee of the Bellvitge Biomedical Research Institute (IDIBELL).
6
Profiling Murine Thymocyte Populations
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