On average, over all experiments, D-ForenRIA needs 12.7 s to detect an action while the basic solution needs around 8 min to detect an action.
Finally, the microbeads were incubated with NaYF4 30%Yb0.5%Tm UCNPs-labeled mouse anti-HSA monoclonal reporter antibody for 30 min to detect the proteins bound on the probe antibody-coated microbeads.
For the −AL, we set the threshold at 2000 nT and the run length at 30 min to detect 92 clusters, which is originally 881 data points over the threshold.
For AU, we set the threshold at 1000 nT and the run length at 30 min to detect 25 clusters, which is originally 226 data points over the threshold.
Records were acquired for at least 1 min to detect mepps.
C-fiber was recorded for 2 min to detect its receptive field and to confirm its conduction velocity (less than 2.0 m/s).
Each cycle consisted of 95°C for l min, 55°C for l min, and 72°C for l min. To detect cell surface expression, cultured human tumor cells were stained with purified anti-human TLR3 antibody (eBioscience, San Diego, CA) or purified Mouse IgG1 control (eBioscience), followed by FITC-conjugated rat anti-mouse IgG1 mAb (clone A85-1, BD PharMingen, San Diego, CA).
Samples were first analyzed by flow cytometry for 1 min to establish a baseline before stimulation with 0.5 µg/ml CXCL12 for 2 min and then 2 µM ionomycin (Sigma) for another 2 min. To detect interaction between CXCR4 and G-proteins, WT, CXCR4-null and ΔT pro-B cells were serum-starved for 2 h at 37°C and then cross-linked with 1 mM DSP (dithio-bis-succinimidyl propionate, Pierce) for 30 min, RT.
Briefly, paraformaldehyde-fixed and permeabilized cells were incubated in 70% ethanol for 10 min followed by 1 m Tris pH 8.0 for 5 min. To detect poly(A)+ mRNA, cells were incubated at 37°C overnight with 1 μ m Cy5-labelled oligo dT 30 probe diluted in hybridization buffer (2× SSC, 25% formamide, 10% dextran sulphate, 0.005% BSA, 1 mg/ml yeast tRNA).
Cells were washed once with PBS (pH 7.4), fixed in 4% formaldehyde for 5 min at room temperature (RT), permeabilized with 0.5% Triton X-100 for 6 min and then blocked with 1% bovine serum albumin (BSA) for 20 min. To detect focal contacts, the cells were incubated with a mouse anti-human vinculin (1∶20, Serotec, Martinsried, Germany) at 4°C overnight.
The cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. To detect and eliminate possible primer dimer artifacts, a dissociation curve was generated by adding a cycle of 95°C for 15 s, 60°C for 1 min and 95°C for 15 s.
