6. Complete 7H9 medium (7H9-OADC-Tw): Add 4.7 g of 7H9
powder and 5 mL of glycerol to 900 mL of deionized water;
autoclave for 10 min at 121
C. Cool to room temperature and
add 100 mL of OADC and 2.5 mL of 20% w/v Tween 80. Add
50 μg/mL hygromycin B or 25 μg/mL kanamycin, as required
(see Note 1).
7. Middlebrook 7H11 agar: Add 21 g of 7H11 powder and 5 mL
of glycerol to 900 mL of deionized water; autoclave for 10 min
at 121
C. Cool to 55
C and add 100 mL of OADC. Add
50 μg/mL hygromycin B or 25 μg/mL kanamycin, as required
(see Note 1).
8. Sterile and ventilated 125 and 250-mL Erlenmeyer.
9. Sterile 50-mL Polypropylene Conical Centrifuge tubes.
10. Sterile 384-well assay plates with lids, compatible with cell
culture and laser-scanning confocal microscope (see Note 2).
11. Laboratory centrifuge to achieve 3750 Â g.
12. Humidified 37
C incubator maintaining a 5% CO 2
atmosphere.
13. 37
C shaking incubator.
14. Microplate fluorescence reader, e.g., Victor 3 (Perkin Elmer)
or any other microplate fluorescence reader, equipped with
emission and excitation filters at 488 and 520 nm, respectively.
2.2 RAW264.7
Macrophage Cell Line
Culture
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. RAW264.7 cells: a murine tumor-derived macrophage cell line
[14] available from the American Type Culture Collection
(ATCC, TIB-71) (see Note 3).
2. Fetal bovine serum (FBS): heat-inactivated at 56
C for 30 min.
Store at À20
C in 50 mL aliquots (see Note 4).
3. Complete RPMI medium (RPMI-FBS): add 50 mL of FBS to a
500 mL bottle of RPMI 1640 medium supplemented with
2 mM glutamine. Store at 4
C for up to 1 month.
4. Dulbecco’s Phosphate-Buffered Saline (DPBS), modified without calcium chloride and magnesium chloride, suitable for cell
culture.
5. Laboratory centrifuge to achieve from 220 up to 330 Â g.
6. Sterile 50-mL Polypropylene Conical Centrifuge tubes.
7. Counting chamber.
8. Humidified 37
C incubator maintaining a 5% CO 2
atmosphere.
9. Dimethylsulfoxide (DMSO): store at room temperature.
High-Content Analysis of Host-Pathogen Interactions
653
powder and 5 mL of glycerol to 900 mL of deionized water;
autoclave for 10 min at 121
C. Cool to room temperature and
add 100 mL of OADC and 2.5 mL of 20% w/v Tween 80. Add
50 μg/mL hygromycin B or 25 μg/mL kanamycin, as required
(see Note 1).
7. Middlebrook 7H11 agar: Add 21 g of 7H11 powder and 5 mL
of glycerol to 900 mL of deionized water; autoclave for 10 min
at 121
C. Cool to 55
C and add 100 mL of OADC. Add
50 μg/mL hygromycin B or 25 μg/mL kanamycin, as required
(see Note 1).
8. Sterile and ventilated 125 and 250-mL Erlenmeyer.
9. Sterile 50-mL Polypropylene Conical Centrifuge tubes.
10. Sterile 384-well assay plates with lids, compatible with cell
culture and laser-scanning confocal microscope (see Note 2).
11. Laboratory centrifuge to achieve 3750 Â g.
12. Humidified 37
C incubator maintaining a 5% CO 2
atmosphere.
13. 37
C shaking incubator.
14. Microplate fluorescence reader, e.g., Victor 3 (Perkin Elmer)
or any other microplate fluorescence reader, equipped with
emission and excitation filters at 488 and 520 nm, respectively.
2.2 RAW264.7
Macrophage Cell Line
Culture
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. RAW264.7 cells: a murine tumor-derived macrophage cell line
[14] available from the American Type Culture Collection
(ATCC, TIB-71) (see Note 3).
2. Fetal bovine serum (FBS): heat-inactivated at 56
C for 30 min.
Store at À20
C in 50 mL aliquots (see Note 4).
3. Complete RPMI medium (RPMI-FBS): add 50 mL of FBS to a
500 mL bottle of RPMI 1640 medium supplemented with
2 mM glutamine. Store at 4
C for up to 1 month.
4. Dulbecco’s Phosphate-Buffered Saline (DPBS), modified without calcium chloride and magnesium chloride, suitable for cell
culture.
5. Laboratory centrifuge to achieve from 220 up to 330 Â g.
6. Sterile 50-mL Polypropylene Conical Centrifuge tubes.
7. Counting chamber.
8. Humidified 37
C incubator maintaining a 5% CO 2
atmosphere.
9. Dimethylsulfoxide (DMSO): store at room temperature.
High-Content Analysis of Host-Pathogen Interactions
653