10. Sterile and ventilated 75 cm
2 and 175 cm
2 -tissue culture flasks
(75 and 175 cm
2 flasks).
11. Versene solution (Gibco) formulated as EDTA solution
(0.48 mM) in PBS: store at 4
C and use within 1 month
after opening.
2.3 THP1
Macrophage Cell Line
Culture
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–11 under Subheading 2.2.
2. THP-1 cells: Human monocyte cell line [15] (ATCC,
TIB-202) (see Note 3).
3. 50 μg/mL Phorbol 12-myristate 13-acetate (PMA) stock solution: dissolve 1 mg of PMA in 20 mL of DMSO. Store
0.1–1 mL aliquots at À20
C in the dark for up to 6 months.
2.4 A549 Human
Type II
Pneumocyte-like Cell
Line Culture
1. See items 2–11 under Subheading 2.2.
2. A549 cells: Human type II pneumocyte-like cell line [16]
(ATCC, CCL-185) (see Note 3).
2.5 Production
of L929-Derived M-CSF
Conditioned Medium
for Murine Primary
Macrophage
Differentiation
and Culture [17]
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–10 under Subheading 2.2.
2. Murine L929 fibroblast cell line (ATCC
® CCL-1™) (see Note
3).
3. Trypsin-EDTA (0.05%) solution (Gibco): store at 4
C and use
within 1 month after opening.
2.6 Production
of J558-Derived
GM-CSF Conditioned
Medium for Murine
Primary Dendritic Cell
Differentiation
and Culture [18]
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–10 under Subheading 2.2.
2. Murine J558 cell line (ATCC
® TIB-6™) (see Note 3).
3. Complete IMDM medium (IMDM-FBS): add 50 mL of FBS
to a 500 mL bottle of IMDM medium supplemented with
50 mM β-mercaptoethanol. Store at 4
C for up to 1 month.
4. 50 mg/mL Geneticin stock solution (G418 disulfate salt).
Store at 4
C for up to 1 year. For J558 cell maintenance,
freshly prepare IMDM-FBS supplemented with 1 mg/mL
Geneticin.
2.7 Murine Primary
Monocyte Isolation
N.B. Requirement of an animal facility and a dedicated laboratory
with a type 2 biosafety cabinet.
1. See items 2–10 under Subheading 2.2.
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Nathalie Deboosere et al.
2 and 175 cm
2 -tissue culture flasks
(75 and 175 cm
2 flasks).
11. Versene solution (Gibco) formulated as EDTA solution
(0.48 mM) in PBS: store at 4
C and use within 1 month
after opening.
2.3 THP1
Macrophage Cell Line
Culture
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–11 under Subheading 2.2.
2. THP-1 cells: Human monocyte cell line [15] (ATCC,
TIB-202) (see Note 3).
3. 50 μg/mL Phorbol 12-myristate 13-acetate (PMA) stock solution: dissolve 1 mg of PMA in 20 mL of DMSO. Store
0.1–1 mL aliquots at À20
C in the dark for up to 6 months.
2.4 A549 Human
Type II
Pneumocyte-like Cell
Line Culture
1. See items 2–11 under Subheading 2.2.
2. A549 cells: Human type II pneumocyte-like cell line [16]
(ATCC, CCL-185) (see Note 3).
2.5 Production
of L929-Derived M-CSF
Conditioned Medium
for Murine Primary
Macrophage
Differentiation
and Culture [17]
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–10 under Subheading 2.2.
2. Murine L929 fibroblast cell line (ATCC
® CCL-1™) (see Note
3).
3. Trypsin-EDTA (0.05%) solution (Gibco): store at 4
C and use
within 1 month after opening.
2.6 Production
of J558-Derived
GM-CSF Conditioned
Medium for Murine
Primary Dendritic Cell
Differentiation
and Culture [18]
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–10 under Subheading 2.2.
2. Murine J558 cell line (ATCC
® TIB-6™) (see Note 3).
3. Complete IMDM medium (IMDM-FBS): add 50 mL of FBS
to a 500 mL bottle of IMDM medium supplemented with
50 mM β-mercaptoethanol. Store at 4
C for up to 1 month.
4. 50 mg/mL Geneticin stock solution (G418 disulfate salt).
Store at 4
C for up to 1 year. For J558 cell maintenance,
freshly prepare IMDM-FBS supplemented with 1 mg/mL
Geneticin.
2.7 Murine Primary
Monocyte Isolation
N.B. Requirement of an animal facility and a dedicated laboratory
with a type 2 biosafety cabinet.
1. See items 2–10 under Subheading 2.2.
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Nathalie Deboosere et al.