E-GEOD-1709 - Transcription profiling of activated or unactivated T cells isolated from peripheral human leukocytes under gravity or vectorless gravity conditions
Submitted on 25 August 2004, released on 25 June 2007, last updated on 27 March 2012
Peripheral blood leukocytes of three human donors (Donors 2, 4, and 5) were isolated from blood bank buffy-coat preparations by Ficoll-Hypaque density gradient centrifugation, and T-cells were purified with high affinity negative selection human CD3+ T-cell enrichment columns. The cells were resuspended in RPMI-1640 with 10% FCS with approximately 3-8 millions cells/mL at room temperature overnight. T-cells were then either unactivated (1g 0 hr), activated (1g 4 hr) with a final concentration of 5 mg/mL Con A + 4 mg/mL anti-CD28 antibody and incubated for 4 hours at 37°C, or loaded onto a RPM rotating at 60°/s (vg). T-cells loaded on the RPM were cultured for 2 hours prior to collection or activation to allow equilibration of cells to the new environment. After incubation, 1 mL of 4 M guanidinium isothiocyanate in 750 mM sodium citrate buffer, pH 7, with N-lauroylsarcosine and ß-mercaptoethanol was added to lyse cells and stabilize RNA.
transcription profiling by array, co-expression, compound treatment, growth condition, replicate, time series
Key gravity-sensitive signaling pathways drive T cell activation. J B Boonyaratanakornkit, A Cogoli, C-F Li, T Schopper, P Pippia, G Galleri, M A Meloni, M Hughes-Fulford. FASEB J 19(14):2020-2022 (2005), Europe PMC 16210397