Procedures Amniotic fluid cell culture A total of three T21 and 5 CN amniocyte samples had been collected by amniocentesis from women at 15 to 21 weeks of gestation, undergoing prenatal diagnosis. These amniotic fluid cells were a fraction with the cells obtained for cytogenetic analysis, and they were grown to confluency in T 12. five cm2 flasks for approximate 10 to 14 days in 50% AmnioMax C100 combined media and 50% Chang Medium D, at the Cytogenetics Laboratory of Mount Sinai Hospital. After chromosomal status was confirmed and every single flask was confluent, we harvested approxi mately 50% of those cells as the initial population for SILAC and placed them in new T 12. five cm2 flasks. Cells from an individual constituted a single sample with no pooling at any step, except for 1,1 mix for SILAC evaluation.
The study protocol was authorized by the Institutional Critique Board of Mount Sinai Hospital. Informed consent was obtained from all participants. The study was performed in accordance with all the Declaration of Helsinki Principles. Steady Isotope Labelling by read full article Amino acids in Cell culture Media Composition SILAC media were ready from customized Dulbecos Modified Eagles Medium devoid in two essen tial amino acids, L arginine and L lysine. Heavy amino acids, L Arg6 and L Lys8, have been supplemented for the medium at a concentra tion of 72 mg L and 90 mg L, respectively, for the heavy medium. For the control medium, amino acids L arginine and L lysine were supplemented at a final concentration of 69 mg L and 85 mg L each and every. Each heavy and light medium had been supplemented with L proline at a concentration of 150 mg L.
All amino acids had been reconstituted in phosphate buffered saline and had been filtered by means of a 0. 22 selleck chemical um filter to receive a sterile solution. Furthermore, 10% of dialyzed FBS and AmnioMAX C100 Sup plement had been added to each heavy and light medium, except for the final 48 hours. Heavy medium was applied to incubate T21 amniocytes, and light medium was utilized to culture CN amniocytes. A mini mum of 5 doubling instances was ensured by culturing cells from half a flask of 12 cm2 surface region to a flask of 175 cm2 surface location at 37 C. Development media had been replaced with fresh media every two to three days over a period of approximately 12 days. When cells turn out to be 90% confluent in a T 175 flask, cells have been rinsed with PBS solution 3 instances, then fresh heavy or light SILAC media were added to the flasks with out FBS or AmnioMAX C100 Supplement. After 48 hours of incu bation, each cells plus the supernatant have been collected and stored at 20 C until use. Cells were harvested with trypsin and washed with PBS before centrifugation. Cells from preliminary experiments had been tested for incorpor ation of the label after five doubling occasions.