Antibiotic/antimycotic solution

Human skin fibroblasts are cultured from skin biopsy samples. The dermis is cut into small pieces (0.5 mm on each side) and placed in a dish containing DMEM, 10% (v/v) FCS, and 1% (v/v) antibiotic-antimycotic solution. When these primary cultures are confluent they are split into novel dishes. Cells between passages three and six are used for experiments. 

Human skin fibroblasts are cultured from skin biopsy samples. The dermis is cut into small pieces (0.5 mm on each side) and placed into a dish in DMEM containing 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic solution. When these primary cultures are confluent they are split and cells between passage three and six are used for experiments. For the cholesterol efflux assay, cells are grown in 24-well plates to 60-80% confluence and are labeled with [1,2-3H]-cholesterol (1 pCi/well) for 24 h. Cells are then washed with DMEM and incubated for 4 h at 37°C with DMEM containing BSA (0.2%, v/v) and either 0 (negative control) or 5-30 pg/ml apoA-I. The efflux medium is collected and centrifuged to remove cell debris. Cells are solubilized in 0.1 mol/1 NaOH and the radioactivity in the efflux media and in the cell lysates is determined by scintillation counting [11, 30, 75]. 

Antibiotic/antimycotic solution (lOOX) from (Gibco) contains... 

Medium 2 (from 4DIV, after transfection) 95 % Neurobasal Medium-A (Gibco ), 2 % B27 supplement 50x (Gibco ), 2 % 100 ruM L-glutamine, 1 % antibiotic/antimycotic solution. 

Culture medium 50 % BME (Sigma Chemicals), 25 % horse serum (Gibco , Life Technologies , Carlsbad, CA), 25 % HBSS (Sigma Chemicals), 0.5 % glucose, 0.5 % 200 mM L-glutamine, 1 % antibiotic/antimycotic solution. 

When the agar has cooled down, add the albumen to the agar solution and swirl gently to mix add 2.4ml of the lOOx antibiotic-antimycotic stock while continuing to mix. Using a sterile 10 ml pipet and pipet aid, distribute 2.5 ml agar-albumen into each petri dish, avoiding the formation of bubbles. Do not dispense more than 2.5 ml, since a thicker substrate will make observation of the embryos more difficult. 

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