Before initiation of the assays, the SMCs were starved into DMEM supplemented with 0.1% fetal bovine serum for 48 hours [ 25, 27].
The cryopreserved PBMC were thawed just prior to the initiation of the assays, diluted into warm RPMI-medium (Gibco Life-Technologies, Grand Island, NY, USA) supplemented with 20% (v/v) foetal calf serum (FCS; Gibco Life-Technologies), and washed twice with RPMI medium.
In most experiments the QIAGEN OneStep RT-PCR Kit (QIAGEN) was used as its hot-start capability allowed convenient on-chip addition of the assay reagents to the microfluidic chip before initiation of the assay.
The dinucleotide initiation of replication assay was performed as described previously [41], [42], and UV cross-linking to model vRNA and cRNA promoters was also performed as reported previously [29], [31], [32].
The sulfated polymers were spiked into the plasma prior to initiation of the assay.
In brief, cells were serum starved for 24 h before initiation of the assay.
Prior to initiation of the assay, cAMP was added to some samples to a final concentration of 30 μM.
Previous work from our group has shown high performance two-dimensional paper-based assays, but the work presented here is particularly significant because all of the necessary reagents were patterned, dried, and stored on a single porous membrane and rehydrated with the addition of sample and buffer upon the initiation of the assay.
In some experiments, either anti-HLA class I (W6/32: mouse IgG2a), anti-HLA-DR (L243: mouse IgG2a), anti-CD4 (NU-TH/I: mouse IgG1), anti-CD8 (NU-TS/C: mouse IgG2a), or anti-CD14 (H14: mouse IgG2a) mAb was added to the wells at a dose of 20 μg ml−1 at the initiation of the assay.
To determine if the increased levels of fabp1a mRNAs was the result of transcriptional initiation, we assayed the levels of hnRNA for these fabp genes in various tissues of zebrafish.
