Primary Schwann cells cultures were obtained from sciatic nerves harvested from adult Sprague Dawley rats. The nerves were collected in cold DMEM plus glutamax (Gibco, Carlsbad, CA, USA) containing 100 U/mL penicillin (R&D system, Minneapolis, MN, USA) and 100 g/mL Streptomycin (R&D system, Minneapolis, MN, USA). Nerves were then gently dissected and the epineurium removed with fine forceps. Fragments of nerves were then plated in 35 mm petri dishes and incubated with glial permissive medium (DMEM plus glutamax, 100 U/mL penicillin and 100 g/mL Streptomycin, 14M forskolin and 100 ng/mL LNRG11; R&D System, Minneapolis, MN, USA) for 2 weeks under culture conditions (37°C, 5% CO2). The medium was replenished every 72 hours. The culture medium was then enriched with 0.125% collagenase type IV (ThermoFisher, Lafayette, CO, USA) and 117 U/mg dispase (Gibco) for 24 hours and mechanically dissociated with a Pasteur glass pipette. The cell suspension was filtered through a 70 µm cell strainer (BD Biosciences) and centrifuged at 100 × g for 5 minutes. The cell pellet was resuspended in glial permissive medium, seeded on 35 mm petri dishes pre-coated with poly-D-lysine (Sigma) and incubated under culture conditions. An antibody complement method was adopted to purify Schwann cells from fibroblasts (Tohill et al., 2004; Kaewkhaw et al., 2012; Pascal et al., 2014).
Streptomycin
Manufactured by R&D Systems
Sourced in United States
Streptomycin is a broad-spectrum antibiotic used in laboratory research settings. It is effective against a wide range of bacterial species, including both Gram-positive and Gram-negative bacteria. Streptomycin functions by inhibiting protein synthesis in bacterial cells, thereby disrupting their ability to grow and replicate.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by R&D Systems (34 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
L glutamine, by R&D Systems (6 mentions)
Recombinant mouse granulocyte macrophage colony stimulating factor, by R&D Systems (4 mentions)
Endothelial growth factor, by R&D Systems (3 mentions)
0.83 m nh4cl buffer, by Merck Group (3 mentions)
M csf, by R&D Systems (3 mentions)
Rankl, by R&D Systems (3 mentions)
Penicillin g, by R&D Systems (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Glutamine, by R&D Systems (2 mentions)
M199 medium, by Thermo Fisher Scientific (2 mentions)
Collagenase 1, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by R&D Systems (2 mentions)
Interleukin 3 (il 3), by R&D Systems (2 mentions)
43 protocols using streptomycin
1
Isolation and Culture of Primary Schwann Cells
2
Differentiation and Culture of Mast Cells
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Mouse bone marrow-derived mast cells (BMMC) were differentiated from the marrow of tibias and femurs of Wt and Ptch1+/− littermate mice and cultured for at 6-8 weeks in RPMI 1640 supplemented with 10% FBS, HEPES (1M), penicillin (100 U/mL), streptomycin, 4 mM L-glutamine (100 μg/mL), sodium pyruvate (1 mM), 2-mercaptoethanol (50 μM), and IL-3 (30 ng/mL, R&D Systems). Cultures were analyzed by flow cytometry and BMMC were identified as FcϵRI and Kit double positive cells as described above. Degranulation assays of BMMC were performed as described (26 (link)).
HMC-1.1 and HMC-1.2 cell lines were cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml). LAD2 cells were cultured in StemPro-34™ supplemented with StemPro-34™ Nutrient Supplement (Gibco), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and SCF (100 ng/ml, R&D Systems).
Primary human mast cell cultures (hMC) were derived from CD34+ lymphocytapheresis progenitors obtained from healthy volunteers following informed consent under a protocol (NCT00001756) approved by the National Institutes of Health Internal Review Board. Cells were cultured as described (27 (link)) and used between 7–9 weeks of culture when >95% were mast cells identified as KIT and FcεRI double positive cells.
HMC-1.1 and HMC-1.2 cell lines were cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml). LAD2 cells were cultured in StemPro-34™ supplemented with StemPro-34™ Nutrient Supplement (Gibco), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and SCF (100 ng/ml, R&D Systems).
Primary human mast cell cultures (hMC) were derived from CD34+ lymphocytapheresis progenitors obtained from healthy volunteers following informed consent under a protocol (NCT00001756) approved by the National Institutes of Health Internal Review Board. Cells were cultured as described (27 (link)) and used between 7–9 weeks of culture when >95% were mast cells identified as KIT and FcεRI double positive cells.
3
Murine Macrophage Differentiation and Activation
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BMDMs were isolated from bone marrow of C57BL/6 wild-type or Caspase-1−/− (Jackson Laboratory) female mice (7–8 weeks old) and differentiated for 7 days in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/ml) and streptomycin (0.1 mg/ml) and 20 ng/ml human M-CSF (R&D Systems). THP-1 (Cell Bank of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI 1640 medium with FBS and penicillin and streptomycin. Phorbol 12-myristate 13-acetate (100 ng/ml, 24 h) was used in differentiation of THP-1 into macrophages. The additives or inhibitors used in in vitro experiments are as follows: purified SAK (0.5 μg/ml), lipopolysaccharide (LPS, 0.2 μg/ml), ATP (5 mM), MCC950 (1 μM, Selleck), potassium channel blocker glibenclamide49 (link) (10 μM, Sigma), KCl (25 mM), NADPH oxidase inhibitor apocynin50 (link) (200 μM, Selleck), lysosome membrane stabilizer dexamethasone51 (link) (200 nM, Sangon Biotech), ROS scavenger NAC52 (link) (1 mM, Sigma), and NF-κB inhibitor BAY 11-7082 (10 μM, Sigma).
4
Plasmacytoid Dendritic Cell Cytokine Regulation
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Isolated pDCs (5 × 104 cells/96-well cavity) were seeded in 200 µl of RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 100 IU ml−1 penicillin, 100 µg ml−1 streptomycin, 2 mM l -glutamine, 10% (vol/vol) human serum and recombinant human (rh) IL-3 [10 ng ml−1; R&D Systems]. To determine the levels of secreted IFNα, pDCs were cultured for 12–14 h in the presence of ODN 2216 (CpG A; 0.26 µM; Thermo Fisher Scientific). To block p75NTR activity, the inhibitory peptide TAT-Pep5 (100 nM, Merck) was added. Recombinant human β-NGF (R&D Systems) was added at the indicated concentrations. The IFNα levels in supernatants were determined using an ELISA (eBioscience). To determine the levels of secreted IL-6, pDCs were stimulated with an antibody specific to human FcεRlα (250 ng ml−1; eBioscience) alone or in the presence of the TAT-Pep5 inhibitor. Recombinant human β-NGF was added at the indicated concentrations. After 14 h, the supernatant was collected to measure IL-6 levels using an ELISA (eBioscience).
5
Chondrocyte Encapsulation in Alginate Beads
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ATDC5 cells were suspended at a density of 2 × 106 cells/ml in a 1.2% solution of sterile alginate (Kelco, Chicago, IL) in 0.15 M NaCl. The cell suspension was slowly expressed through a 22-gauge needle dropwise into a 100 mM calcium chloride solution. Beads were allowed to polymerize in this solution for 10 min before two consecutive washes with 0.15 M NaCl followed by two washes in DMEM/F12. After washing, beads containing ATDC5 cells were then placed into 12-well culture plates (10 beads/well) in 2 ml of DMEM/F12 containing 5% FBS, penicillin (100 μg/mL)-streptomycin (50 μg/mL), insulin-transferrin-selenium, recombinant human bone morphogenetic protein (BMP)-2 (0.25 μg/mL, R&D Systems, Minneapolis MN), and 50 μg/ml ascorbic acid. ATDC5 cells were cultured in alginate beads with the growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. Medium was changed once in three days. Medium was then removed from individual wells, and the beads were dissolved by incubation for 10–15 min at 4 °C in dissolving solution (55 mM sodium citrate, 30 mM Na2EDTA, 0.15 M NaCl, pH 6.8). The resulting suspension was centrifuged at 900 rpm for 10 min at 4 °C to separate the ATDC5 cells.
6
Establishing Primary and Cell Line Cultures
7
Osteogenic Differentiation of Rat and Human MSCs
8
Osteoclast Differentiation from Spleen Cells
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Spleen cells were isolated from 4- to 6-week-old WT or src−/− mice and cultured under 5% CO2 at 37 °C in alpha modification of Eagle's minimum essential medium from Sigma-Aldrich supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) with human macrophage colony-stimulating factor (30 ng/ml) from R&D Systems Inc for 3 days, and then, the cells (100,000 cells/well, in a 24-well plate) were replated on culture plates from Corning, coverslips (Fisher Scientific), or dentin slices and cultured with human soluble RANKL (100 ng/ml) from R&D Systems Inc for 5 days. Cells were infected with adenoviruses expressing cre recombinase (CRE) (multiplicity of infection [MOI] 50) and Src family kinases or chimeras (MOI 50) on day 4 after replating and culture for 1 day.
9
Isolation and Culture of HUVECs
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Primary HUVECs were collected from human umbilical veins by digestion in 0.1% collagenase I in M199 medium (Gibco, NY, USA) for 15 min. The solution was collected and centrifuged at 1000 rotations per minute (rpm) for 10 min. The cells were cultured in M199 medium that contained 10% fetal bovine serum (Gibco, NY, USA), 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 20 ng/ml endothelial growth factor (R&D, Minneapolis, MN, USA) in an incubator at 37°C and 5% CO2. HUVECs from passage 3 were used in the present study.
10
Isolation and Culture of Bone Marrow-Derived Dendritic Cells
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Bone marrow-derived DCs (BMDCs) were isolated from the bone marrow cells of C57BL/6 (H-2b) mice.26 (link) Briefly, bone marrow was collected from the tibias and femurs by flushing with RPMI 1640 using a 1 mL syringe with a 26 G needle (BD Biosciences, San Jose, CA, USA). After one wash (490×g, 5 minutes) in RPMI 1640 medium, the cells were incubated with 5 mL of 0.83 M NH4Cl buffer (Sigma-Aldrich) to lyse red blood cells at 37°C for 5 minutes. Then, cells were centrifuged twice in RPMI 1640 medium at 150×g. The bone marrow cells (2×106 cells) were collected and cultured in 100 mm petri dishes (BD, Franklin Lakes, NJ, USA) containing 10 mL of the RPMI 1640 medium supplemented with 10% of heat-inactivated fetal bovine serum, 50 IU/mL penicillin, 50 mg/mL streptomycin, and 20 ng/mL mouse recombinant granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA). After 7 days, nonadherent and loosely adherent cells (immature DCs) were harvested, washed, and used for in vitro experiments.
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