Finally, the last method concentrates on the multicellular stage
of D. discoideum and how the developmental cycle affects the outcomes of infection. The multicellular stage of D. discoideum usually
assembles hundreds of thousands of cells [19]. Monitoring the
infection during the transition from single autonomous cells to an
ordered multicellular entity represents a challenge. To visualize and
quantitate the fates of both host and pathogen in a controlled
manner, cells are sorted by fluorescence-assisted cell sorting
(FACS) to adjust the proportions of infected cells in the mix. The
developmental cycle is then imaged live in large fields using tile
scans and different channels. This chapter illustrates some aspects of
this process by computing the colocalization of bacteria and host.
2 Materials
2.1 Cell Culture
1. D. discoideum laboratory strain Ax2(Ka) [20] (the strains can
be obtained from the stock center, http://www.dictybase.org/
StockCenter/StockCenter.html, ID: DBS0235521).
2. D. discoideum laboratory strain Ax2-214 [21] (StockCenter,
ID: DBS0235534).
3. D. discoideum laboratory strain Ax2(Ka), transformed with
GFP-LifeAct [22], resistant to hygromycin.
4. HL5c medium including glucose supplemented with vitamins
and microelements (ForMedium): resuspend 26.55 g of powder in 1 L of deionized water. Filter-sterilize (Steritop 0.22 μm
filters, Millipore) (see Note 1).
5. Penicillin/streptomycin 100Â stock solution: 10,000 U and
10,000 μg/mL, respectively.
6. Hygromycin: Prepare a 1000Â stock solution by dissolving
100 mg of powder in 1 mL deionized water. Filter-sterilize
and store at À20
C.
7. 10 cm cell culture dishes, tissue culture-treated polystyrene.
2.2 Mycobacterial
Culture (see Note 2)
1. M. marinum M strain (ATCC BAA-535).
2. M. marinum M strain variants, GFP-expressing, e.g., pmsp12::
GFP; kanamycin resistant [23], or Cherry-expressing
(pCherry10; hygromycin resistant, Addgene #24664).
3. 7H9 medium (Difco): dissolve 4.7 g of Middlebrook 7H9
powder in 900 mL of deionized water. Add 500 μL of Tween
80 and 2 mL of glycerol and autoclave. Let the medium cooldown and add 10% v/v of Middlebrook OADC supplement
(Becton Dickinson). Filter-sterilize and store at 4
C.
4. Kanamycin: prepare a 1000Â stock solution by dissolving
30 mg of powder in 1 mL deionized water. Filter-sterilize and
store at À20
C.
186
Manon Mottet et al.
of D. discoideum and how the developmental cycle affects the outcomes of infection. The multicellular stage of D. discoideum usually
assembles hundreds of thousands of cells [19]. Monitoring the
infection during the transition from single autonomous cells to an
ordered multicellular entity represents a challenge. To visualize and
quantitate the fates of both host and pathogen in a controlled
manner, cells are sorted by fluorescence-assisted cell sorting
(FACS) to adjust the proportions of infected cells in the mix. The
developmental cycle is then imaged live in large fields using tile
scans and different channels. This chapter illustrates some aspects of
this process by computing the colocalization of bacteria and host.
2 Materials
2.1 Cell Culture
1. D. discoideum laboratory strain Ax2(Ka) [20] (the strains can
be obtained from the stock center, http://www.dictybase.org/
StockCenter/StockCenter.html, ID: DBS0235521).
2. D. discoideum laboratory strain Ax2-214 [21] (StockCenter,
ID: DBS0235534).
3. D. discoideum laboratory strain Ax2(Ka), transformed with
GFP-LifeAct [22], resistant to hygromycin.
4. HL5c medium including glucose supplemented with vitamins
and microelements (ForMedium): resuspend 26.55 g of powder in 1 L of deionized water. Filter-sterilize (Steritop 0.22 μm
filters, Millipore) (see Note 1).
5. Penicillin/streptomycin 100Â stock solution: 10,000 U and
10,000 μg/mL, respectively.
6. Hygromycin: Prepare a 1000Â stock solution by dissolving
100 mg of powder in 1 mL deionized water. Filter-sterilize
and store at À20
C.
7. 10 cm cell culture dishes, tissue culture-treated polystyrene.
2.2 Mycobacterial
Culture (see Note 2)
1. M. marinum M strain (ATCC BAA-535).
2. M. marinum M strain variants, GFP-expressing, e.g., pmsp12::
GFP; kanamycin resistant [23], or Cherry-expressing
(pCherry10; hygromycin resistant, Addgene #24664).
3. 7H9 medium (Difco): dissolve 4.7 g of Middlebrook 7H9
powder in 900 mL of deionized water. Add 500 μL of Tween
80 and 2 mL of glycerol and autoclave. Let the medium cooldown and add 10% v/v of Middlebrook OADC supplement
(Becton Dickinson). Filter-sterilize and store at 4
C.
4. Kanamycin: prepare a 1000Â stock solution by dissolving
30 mg of powder in 1 mL deionized water. Filter-sterilize and
store at À20
C.
186
Manon Mottet et al.