CII-specific T cells from draining LNs were enumerated and characterized using a DR1-CII tetramer [28 (link)]. Briefly, soluble DR1 covalently linked to the immunodominant CII peptide was produced in S2 cells, affinity-purified, biotinylated by BirA (Avidity, Aurora, CO, USA), and formed into multimeric units by stepwise addition of PE-labeled streptavidin (Rockland Immunochemicals, Limerick, PA, USA). For identification of CII-specific T cells ex vivo, LN cells were incubated with 1 μg of the tetramer at 37 °C for 2.5 h in complete HL-1 medium (50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate, 0.05 mM β-mercaptoethanol, 2 mM l -glutamine, and 0.1 % bovine serum albumin; BioWhittaker/Lonza, Walkersville, MD, USA) supplemented with 5 mM NaN3. At the end of the incubation, antibodies specific for various CD markers were added and cells were incubated for an additional 30 minutes at 4 °C. Samples were then washed and resuspended in PBS supplemented with 0.1 % NaN3 and 2 % fetal bovine serum and analyzed by flow cytometry (BD LSR II). DR1-restricted, CII257–274-specific and HA306–318-specific T-cell hybridomas were used as positive and negative controls, respectively, to monitor the specificity and sensitivity of the DR1-CII tetramer (see Additional file 1 ).
Penicillin g sodium
Manufactured by Lonza
Sourced in Switzerland
Penicillin G sodium is a white to off-white crystalline powder. It is a commonly used antibiotic agent.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin sulfate, by Lonza (3 mentions)
Streptomycin sulphate, by Lonza (3 mentions)
Amphotericin b, by Lonza (3 mentions)
L glutamine, by Lonza (2 mentions)
Foetal calf serum, by Capricorn (2 mentions)
Sk es 1, by Leibniz Institute DSMZ (2 mentions)
Qpcr mycoplasma test kit, by AppliChem (2 mentions)
Lsr 2, by BD (1 mentions)
Bovine serum albumin (bsa), by Lonza (1 mentions)
β mercaptoethanol, by Lonza (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Low molecular weight chitosan, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Citric acid monohydrate, by Honeywell (1 mentions)
2 2 6 6 tetramethylpiperidine 1 oxyl radical, by Merck Group (1 mentions)
Naclo, by Merck Group (1 mentions)
Rat tail collagen, by Merck Group (1 mentions)
A673 cells, by Merck Group (1 mentions)
7 protocols using penicillin g sodium
1
Enumeration and Characterization of CII-Specific T Cells
2
Cell Culture Conditions Optimization
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The RPMI-1640 medium was used for culturing the Caco-2, MCF7, and mice splenocytes. For the HepG2 cell line, cells were cultured in DMEM. All the media were supplemented with 10% fetal bovine serum (FBS), 2 mM l -glutamine, containing 100 units per mL penicillin G sodium, 100 units per mL streptomycin sulphate, and 250 ng per mL amphotericin B. All from Lonza (Basel, Switzerland). Cells were maintained at sub-confluency at 37 °C in humidified air containing 5% CO2. For sub-culturing, monolayer cells were harvested after trypsin/EDTA treatment at 37 °C. Cells were used when confluence had reached 75%.
3
Biocompatible Risedronate-Chitosan Hydrogel
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Bleached bagasse Kraft Pulp was kindly delivered by Qena Paper Industry Company, Egypt. The chemical composition of bagasse pulp was 71% α-cellulose, 30% pentosans, 0.8% ash, and a degree of polymerization (DP) of 1200. Risedronate sodium was kindly supplied by Hikma Pharmaceutical and Chemical Industries Company, Egypt. Chitosan low molecular weight, NaOH, urea, NaBr, NaClO, 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) were purchased from Sigma-Aldrich, Germany. Citric acid monohydrate was purchased from Riedel-deHaen, Germany. Fetal bovine serum (FBS), L-glutamine, penicillin G sodium, streptomycin sulfate, and amphotericin B were obtained from Lonza, Basel, Switzerland. MTT (3-[4, 5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide), acridine orange and ethidium bromide were obtained from Merck, Darmstadt, Germany.
4
Cell Culture Protocol for Sarcoma Cell Lines
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WE-68 cells were a gift from Dr F. van Valen (Münster, Germany). SK-ES-1 and HeLa cells were purchased from the DSMZ (Braunschweig, Germany). A673 cells were purchased from Sigma Aldrich (Deisenhofen, Germany). WE-68, SK-ES-1 and HeLa cells were cultured in RPMI 1640 medium and A673 cells were cultured in DMEM (Lonza, Cologne, Germany). Media were supplemented with 10% foetal calf serum (Capricorn Scientific, Ebsdorfergrund, Germany), 2 mM l -glutamine, 100 units/ml penicillin G sodium and 100 µg/ml streptomycin sulphate (Lonza). All tissue culture vessels used for the cultivation of ES cells were coated with rat tail collagen (Merck, Darmstadt, Germany) at a concentration of 5 µg/cm2. Cells were maintained at a temperature of 37 °C in a humidified 5% CO2 incubator and routinely passaged at a confluence of ~ 90%. Cells were tested to be negative for mycoplasma with the qPCR Mycoplasma Test Kit from Applichem (Darmstadt, Germany).
5
Cell Culture of Sarcoma Cell Lines
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WE-68 (RRID: CVCL_9717) cells were kindly provided by Dr. F. van Valen (Münster, Germany), SK-ES-1 (RRID: CVCL_0627) cells were purchased from the DSMZ (Braunschweig, Germany), and A673 (RRID: CVCL_0080) cells were purchased from Sigma Aldrich (Deisenhofen, Germany). WE-68 and SK-ES-1 cells were cultured in RPMI 1640 medium, and A673 cells were cultured in DMEM (Capricorn Scientific, Ebsdorfergrund, Germany). Media were supplemented with 10% foetal calf serum (Capricorn Scientific), 100 units/ml penicillin G sodium and 100 µg/ml streptomycin sulphate (Lonza, Basel, Switzerland). Cells were cultured throughout in rat-tail collagen-coated (5 µg/cm2; Merck, Darmstadt, Germany) cell culture vessels. Cells were maintained in an incubator at 37 °C and 5% CO2 and regularly passaged at a confluence of 90%. Mycoplasma contamination was tested with the qPCR Mycoplasma Test Kit from Applichem (Darmstadt, Germany).
6
Caco-2 and LS513 Cell Culture Protocol
7
Cytotoxicity Evaluation of Zinc Sulfate
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Zinc sulfate (ZnSO4·7H2O) was purchased from Sigma, Aldrich, Germany, and was used for zinc solution preparation. Nutrient broth and agar medium were purchased from Oxoid, UK. 3-(4, 5-dimethyIthiazol-2-y1)-2-5-diphenyl tetrazolium bromide (MTT) was obtained from Merck KGaA (Darmstadt, Germany). 10% fetal bovine serum (FBS), 2 mM L-glutamine solution, 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulfate, and 250 g/ml amphotericin B were obtained from Lonza, Basel, Switzerland. All chemicals were analytical grades.
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