text: Printing solution
  Several transfection reagents were tested (data not shown), and we found that the X-tremeGENE siRNA transfection reagent (Roche) gave good transfection efficiencies both for plasmids and siRNAs, and chose to use this reagent for all transfected cell microarray experiments in the present study. For printing the arrays, one major challenge is to find a good balance between high transfection efficiency and spatially confined spots to avoid cross-contamination between the spots. In order to optimize the reverse transfection protocol for HEK 293ind-ICER IIgamma cells (see below) and X-tremeGENE transfection reagent, we investigated the effect of varying the concentrations of gelatine and sucrose in the printing solution. These reagents have been reported to influence both the transfection efficiency and spot integrity (6,27). Sucrose was observed to be specifically beneficial for obtaining high transfection efficiency when storing the arrays for several weeks before use (data not shown). Figure 1A and B show representative images of the observed effects of varying the concentrations of gelatine and sucrose. We observed that the transfection efficiency increased with increasing gelatine concentration (tested in the range 0.01-0.40%). However, an increased disturbance of the spatial definition of the spots was observed with increasing concentrations of gelatine or sucrose (tested in the range 0-100 mM). A combined effect of the concentrations of gelatine and sucrose was also observed, as low concentrations of gelatine allowed us to use higher concentrations of sucrose than with higher concentrations of gelatine before cells spread outside the spots. Based on several optimizing experiments, we found that 3 microl X-tremeGENE solution per microgram nucleic acid, 25 mM sucrose and 0.1% gelatine in the final printing solution reproducibly gave spatial restricted transfection with high transfection efficiency printing the arrays with both a pipette tip and a hand-held arrayer (see below). 
  Figure 1.
  Effects of sucrose and gelatine concentrations on spot integrity and transfection efficiency. (A) Array printed with pDsRed (50 ng/microl) in a printing solution with different gelatine and sucrose concentrations. Scanning image of the whole array and magnifications of specific spots. (B) Array printed with pEGFP (50 ng/microl) in a printing solution with 25 mM sucrose and four different concentrations of gelatine. Top: Box plot of the fluorescence intensities in each spot (n = 32-34). Bottom: Scanning image showing squares of seven times five spots for the four gelatine concentrations. From left to right: 0.01, 0.05, 0.1 and 0.2% gelatine. The DNA-lipid-gelatine-sucrose solutions were printed manually with a 10 microl pipette tip (A) or by MicroCasterTM manual arrayer system (B).
  In a 1.5 ml microcentrifuge tube, plasmid (1 microg/microl) and siRNA were mixed with growth medium without fetal calf serum (FCS), 0.5 microl 1.5 M sucrose and 3 microl X-tremeGENE per microgram nucleic acid to a final volume of 22.5 microl. After 15-20 min of incubation, 7.5 microl 0.4% gelatine (Type B, G9391, Sigma) was added to give 30 microl printing solution. The gelatine solution was prepared as described by Ziauddin and Sabatini (1). To achieve sufficient level of expression from the transfected plasmids, 25-50 ng/microl pEGFP-N1 or pDsRed-express-N1 and 50-75 ng/microl of CRE or NFkappaB reporter plasmids was used. For siRNA studies, 2-30 ng/microl siRNA in the final printing solution was used.
  
entities.text: ['cell']
entities.type: Cell
entities.entity_id: PMC-2528170-sec-05_T2
entities.text: ['HEK 293ind-ICER IIgamma cells']
entities.type: Cell
entities.entity_id: PMC-2528170-sec-05_T3
entities.text: ['cells']
entities.type: Cell
entities.entity_id: PMC-2528170-sec-05_T4
entities.text: ['serum']
entities.type: Organism_substance
entities.entity_id: PMC-2528170-sec-05_T6
entities.text: ['fetal']
entities.type: Developing_anatomical_structure
entities.entity_id: PMC-2528170-sec-05_T1
entities.text: ['plasmids']
entities.type: Cellular_component
entities.entity_id: PMC-2528170-sec-05_T5
entities.text: ['plasmids']
entities.type: Cellular_component
entities.entity_id: PMC-2528170-sec-05_T7