2. 6- to 10-weeks old C57BL6 mice (used to collect bone marrow
and purify murine primary monocytes).
3. 10Â Red blood cell lysis buffer: add 15.6 g of NH 4 Cl, 2 g of
NaHCO 3 and 74 mg of EDTA in 200 mL of deionized water.
Adjust the pH to 7.3 using 12 M HCl and filter-sterilize
through a 0.22 μm cellulose ester membrane. Store 5 mL
aliquots at À20
C for up to 1 year. For cell preparation: freshly
prepare 1Â Red blood cell lysis buffer by diluting 5 mL of the
10Â solution with 45 mL of deionized water. Store at 4
C.
2.8 Culture of Murine
Bone Marrow-Derived
Macrophages (BMDM)
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–11 under Subheading 2.2.
2. Murine primary monocytes purified from 6- to 10-weeks old
C57BL6 mice bone marrow (see Subheading 2.7, items 1–3,
and Note 5).
3. Murine Macrophage-Colony Stimulating Factor (M-CSF)
stock (0.1 mg/mL): resuspend 100 μg of lyophilized M-CSF
in 1 mL of sterile deionized water. Store at À20
C in 100 μL
aliquots (see Note 6). Avoid more than 3 freeze/thaw cycles.
2.9 Culture of Murine
Bone
Marrow-Dendritic
Cells (BMDC)
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–8 under Subheading 2.2.
2. Murine hematopoietic stem cells purified from 6- to 10-weeks
old C57BL6 mice bone marrow (see Subheading 2.7 items 1–
3, and Note 5).
3. Murine Granulocyte Macrophage-Colony Stimulating Factor
(GM-CSF) stock (0.1 mg/mL): resuspend 100 μg of lyophilized GM-CSF in 1 mL of sterile deionized water. Store at
À20
C in 100 μL aliquots (see Note 7). Avoid more than
3 freeze/thaw cycles.
4. 0.5 M EDTA solution (Sigma-Aldrich): store at 4
C for up to
3 years. For cell preparation, freshly prepare 2 mM EDTA
solution from the 0.5 M stock solution with pre-warmed
(37
C) DPBS, no calcium, no magnesium.
5. 2 mM EDTA solution. For cell preparation, freshly prepare
2 mM EDTA solution by dissolving 29.2 mg of EDTA in
50 mL of DPBS. Filter-sterilize through a 0.22 μm PTFE
membrane and pre-heat up to 37
C.
6. Sterile 78.5 cm (100 Â 20 mm) and 500 cm
(245 Â 245 Â 25 mm) square culture petri dishes.
High-Content Analysis of Host-Pathogen Interactions
655
and purify murine primary monocytes).
3. 10Â Red blood cell lysis buffer: add 15.6 g of NH 4 Cl, 2 g of
NaHCO 3 and 74 mg of EDTA in 200 mL of deionized water.
Adjust the pH to 7.3 using 12 M HCl and filter-sterilize
through a 0.22 μm cellulose ester membrane. Store 5 mL
aliquots at À20
C for up to 1 year. For cell preparation: freshly
prepare 1Â Red blood cell lysis buffer by diluting 5 mL of the
10Â solution with 45 mL of deionized water. Store at 4
C.
2.8 Culture of Murine
Bone Marrow-Derived
Macrophages (BMDM)
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–11 under Subheading 2.2.
2. Murine primary monocytes purified from 6- to 10-weeks old
C57BL6 mice bone marrow (see Subheading 2.7, items 1–3,
and Note 5).
3. Murine Macrophage-Colony Stimulating Factor (M-CSF)
stock (0.1 mg/mL): resuspend 100 μg of lyophilized M-CSF
in 1 mL of sterile deionized water. Store at À20
C in 100 μL
aliquots (see Note 6). Avoid more than 3 freeze/thaw cycles.
2.9 Culture of Murine
Bone
Marrow-Dendritic
Cells (BMDC)
N.B. Requirement of a dedicated laboratory with a type 2 biosafety
cabinet.
1. See items 2–8 under Subheading 2.2.
2. Murine hematopoietic stem cells purified from 6- to 10-weeks
old C57BL6 mice bone marrow (see Subheading 2.7 items 1–
3, and Note 5).
3. Murine Granulocyte Macrophage-Colony Stimulating Factor
(GM-CSF) stock (0.1 mg/mL): resuspend 100 μg of lyophilized GM-CSF in 1 mL of sterile deionized water. Store at
À20
C in 100 μL aliquots (see Note 7). Avoid more than
3 freeze/thaw cycles.
4. 0.5 M EDTA solution (Sigma-Aldrich): store at 4
C for up to
3 years. For cell preparation, freshly prepare 2 mM EDTA
solution from the 0.5 M stock solution with pre-warmed
(37
C) DPBS, no calcium, no magnesium.
5. 2 mM EDTA solution. For cell preparation, freshly prepare
2 mM EDTA solution by dissolving 29.2 mg of EDTA in
50 mL of DPBS. Filter-sterilize through a 0.22 μm PTFE
membrane and pre-heat up to 37
C.
6. Sterile 78.5 cm (100 Â 20 mm) and 500 cm
(245 Â 245 Â 25 mm) square culture petri dishes.
High-Content Analysis of Host-Pathogen Interactions
655