hybridization gasket slide—8 microarrays per slide format  (Agilent technologies)

Journal: Journal of Leukocyte Biology

Article Title: Prolyl-4-hydroxylase domain 3 (PHD3) is a critical terminator for cell survival of macrophages under stress conditions

doi: 10.1189/jlb.2HI1013-533R

Figure Lengend Snippet: (A) HIF-1α, HIF-2α, PHD2, and PHD3 protein levels were analyzed after cultivating BMDM for 24 h at 20% or 1% O2. (B) The expression of HIF-1 target genes, such as PKM2, Pfk, and PDK-1, on mRNA level was analyzed after cultivating BMDM for 24 h at 20% O2 or 1% O2 using qRT-PCR. (C) PHD3−/− [knockout (KO)] and WT BMDM were cultivated for 48 h at 20% O2 and 1% O2. Subsequently, cells were lysed and RNA isolated. RNA samples were analyzed in a transcriptome microarray assay. The volcano blots were generated based on the microarray results. (D) PHD3−/− and WT BMDM were cultured with or without serum for 24 h or stimulated with 100 ng LPS for 12 h. Subsequently, NF-κB activities were analyzed. As a positive control for the NF-κB activity assay, a cell extract of HeLa cells, which were treated with LPS, was used; n = 3; mean ± sd; **P < 0.01, and ***P < 0.001. rel. LU, Relative light units.

Article Snippet: The hybridization was then carried out on the Agilent microarray hybridization slide at 65°C for 17 h. All subsequent steps were followed as instructed by the Low Input Quick Amp Labeling protocol.

Techniques: Expressing, Quantitative RT-PCR, Knock-Out, Isolation, Microarray, Generated, Cell Culture, Positive Control, Activity Assay