RNA concentration and purity was measured within a Nanodrop spectrophot ometer, 200 ng of RNA were reverse transcribed using the SuperScript 1st Strand Synthesis Method and cDNA was utilised for TaqMan Gene Expres sion assays particular for p21 and beta 2 microglobulin, Serious time PCR evaluation was carried out on a light cycler 480, Parabolic Flight experiments The scientific local community in Europe shares just one lar ger aircraft that is licensed for parabolic manoeuvres, an Airbus A300 developed in 1973, which is operated from the Bordeaux M?rignac airport or from Cologne airport through the company Novespace and is flying as much as 9 campaigns each 12 months, Experi ments have been carried out throughout the 8th, 9th,10th and 13th DLR Parabolic Flight Campaign and through the 45th ESA Parabolic Flight Campaign inside the Air bus A300 ZERO G, a specially configured check aircraft, and under the stand ing orders of NOVESPACE and the CEV, Improvement of in flight hardware For the in flight experiments on board the Airbus A300 ZERO G, we designed an experimental procedure, which will allow cell culture exams with residing mammalian cells in microgravity.
Throughout the in depth layout from the major and secondary functional aspects the essential style and design, principles easy, clear and risk-free had been met. Primary value was positioned on realising the direct safety procedure a cool way to improve dur ing the growth activity. The experimental structure includes 3 experiment racks, All modules have already been produced for doing experiments with living mammalian cells in the course of parabolic flights and make it possible for storage of cell culture compartments till the get started in the experiment, injec tion of the fluid at the starting of every para bola and automated injection of a 2nd fluid following 20s with the end of each parabola.
Suitable in flight our site controls have already been obtained through the 1 g flight phase directly prior to or right after the parabola. The module procedure can accommodate 60 cell culture containers for every flight. Injection of all fluids operates automatically and is pre programmed, though exchange of cell culture containers and supervision of the experiment is completed from the researchers throughout the parabolic flight. For a techni cal description of your in flight hardware, please refer to your supplemental file 1, Cell culture and planning for parabolic flights Human Jurkat T cells have been cultured in RPMI 1640 medium, supple mented with 10% FCS and anti biotics. PBMCs have been isolated in the buffy coat fraction from voluntary nutritious donors working with Ficoll Hypaque den sity gradient centrifugation. CD4 T lymphocytes were isolated from PBMCs utilizing a commercially out there MACS separation process, For each experiment, a separate blood donor was made use of. In flight ug and 1 g manage experiments have been per formed in distinctive in flight cell culture bags containing 3 107 cells in 15 ml medium per bag.