Gentamycin

Manufactured by Miltenyi Biotec

Gentamycin is a broad-spectrum antibiotic used in cell culture media to prevent bacterial contamination. It is effective against a variety of gram-positive and gram-negative bacteria.

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2 protocols using gentamycin

Tissue Processing Protocol for Flow Cytometry

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Several solutions were used in sample processing. Collection medium consisted of 50 µg/ml gentamycin (Sigma-Aldrich), 2% Penicillin-Streptomycin (P/S), 12.5 µg/ml Fungizone (Amphotericin B, Gibco) and 20% fetal calf serum (FCS) (Bodego) in RPMI 1640 (Gibco). Digestion medium consisted of 30 IU/ml collagenase IV (Worthington), 1% FCS and 12.5 µg/ml DNAse (Roche) in IMDM (Gibco). Washing medium consisted of RPMI 1640 supplemented with 2% FCS and 2% P/S. FACS buffer contained 2% FCS and 2 mM EDTA in PBS. Red blood cell lysis buffer consisted of 155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA (pH 7.4) in PBS. T cell culture medium consisted of 5% human serum (Sanquin), 5% FCS, 50 µg/ml gentamycin and 1.25 µg/ml Fungizone in 20/80 T cell mixed media (Miltenyi Biotech). Freezing medium consisted of 10% DMSO (Corning) and 30% FCS in IMDM.

van Asten S.D., de Groot R., van Loenen M.M., Castenmiller S.M., de Jong J., Monkhorst K., Haanen J.B., Amsen D., Bex A., Spaapen R.M, & Wolkers M.C. (2021). T cells expanded from renal cell carcinoma display tumor-specific CD137 expression but lack significant IFN-γ, TNF-α or IL-2 production. Oncoimmunology, 10(1), 1860482.

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Generating Mature Dendritic Cells

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Peripheral blood mononuclear cells (PBMCs) from human healthy donors were thawed from ImmunXperts SA (Belgium) biobank. Monocytes were isolated from PBMCs using a MACS magnetic separation column CD14 MicroBeads (Miltenyi) and purity was evaluated by CD14 FACS staining (Fortessa). Cells were then resuspended at a cellular density of 10⁶ cells/mL and plated into a 24-well tissue culture microplate (1mL per well) in CellGenix DC medium (CellGenix, Cat.N° 20801-0500) added with Gentamycin, IL-4 (Miltenyi, 130-093-866) and GM-CSF (Miltenyi, 130-093-922) for 5 days. At day 5, cells were stained for FACS analysis with several DC activation markers to assess their immature dendritic cell (iDC) state: CD14-FITC (Miltenyi, 130-110-518), CD40-BV510 (BD Biosciences, 563456), CD80-BV421 (BD Biosciences, 564160), CD83-PE-Vio 770 (Miltenyi, 130-110-505), CD86-APC (Miltenyi, 130-116-161), CD209-PE (Miltenyi, 130-117-706), and HLA-DR-BUV395 (BD Biosciences, 564040). On the same day, respective antigens (10µg/mL) were added to the cell culture for 48h. At day 7, cells were stained for FACS analysis with same markers to assess their mature state. LPS (Sigma, L4391-1MG) (1µg/mL), and TNF (Miltenyi, 130-094-024) (800U/mL), with IL-1b (Miltenyi, 130-093-898) (150U/mL), were used as positive control.

García-Bengoa M., Vergara E.J., Tran A.C., Bossi L., Cooper A.M., Pearl J.E., Mussá T., von Köckritz-Blickwede M., Singh M, & Reljic R. (2023). Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice. Frontiers in Immunology, 14, 1307429.

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