TLR7-rsq
Ontology highlight
ABSTRACT:
The spleens were homogenized in a gentleMACSM-^Y Dissociator (Miltenyi Biotech, Germany). For red blood cell lysis the cells were resuspended in 3ml ACK lysis buffer (Sigma) and incubated for 3 min at RT. 7ml Medium (RPMI/2.5% FCS/1%PSG/1xbeta-mercaptoethanol were added. The cells were washed 1x in medium and resuspended in 5ml. 6x10^5 cells were treated with the following compounds for 4h. (final volume of 500 ul):
Treatments: R0006 (5 ul/10E+06 cells + 5 ul DOTAP), DOTAP (5 ul + 5ul water x2 replicates), 10 uM R848 and DMSO.
RNA extracted using QUIAShredder/RNeasy columns and stored at -80C until labeling was performed. Labeling was caried out following Agilent QUICKAMP 2-color labeling protocol. Briefly, 400 ng RNA was retrotranscribed and labeled using cyanine3 (reference) or Cyanine5 (treatment) and purified with RNeasy columns. Yield and efficiency of dye incorporation were measured at NanoDrop. Only reactions with yields >0.825 ug cRNA and yields >8 pmol dye/ug cRNA were used for hybridization.
Cy5 labeling: R848, R0006
Cy3 labeling: DOTAP, DMSO
ORGANISM(S): Mus musculus
SUBMITTER: Igor Leykin
PROVIDER: E-MEXP-3051 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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