3. Prepare transfection reaction by mixing together DPBS and
transfection reagent (Lipofectamine LTX, RNAiMAX or
HiPerfect Transfection Reagent). Vortex briefly. Refer to
Table 1 for the appropriate transfection reagent and volumes
needed for each cell type.
4. Incubate 3 min at room temperature.
5. Distribute 10 μL of the transfection reaction per well for each
plate using an automatic plate dispenser (see Note 10) or a
multichannel pipette.
6. Incubate 20 min at room temperature.
7. Prepare cells as required (see Subheadings 3.2–3.4 and 3.7–
3.10) in RPMI-FBS, containing 40 ng/mL M-CSF, GM-CSF
or hM-CSF for primary murine macrophages, murine dendritic
cells and human macrophages, respectively, or no growth factors for cell lines.
8. Add the appropriate number of cells in 20 μL per well
(Table 1).
9. Incubate 6 h at 37
C, 5% CO 2 .
Automated image acquisition and image-based analysis
Cell staining
a
b
c
1. Reverse transfection
a. siRNA
b. Transfection
reagent
c. Cells
1. Compounds
distribution
2. Infection of
cells in
suspension
2. Cells
distribution
1. Mutants
distribution
2. Infection of adherent cells
5 daysincubation
Fig. 2 Overall scheme for the different assays described in this chapter
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