2.10 Human Primary
Cell Purification
and Culture
N.B. Requirement of a type 2 biosafety cabinet in a dedicated
biosafety laboratory adapted for pathogens of class 2.
1. See items 2–9 under Subheading 2.2.
2. Healthy volunteer peripheral blood samples.
3. Ficoll’s density gradient solution (density ¼ 1.077). Store at
room temperature in the dark.
4. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS): add 5 mL of FBS in a 500 mL DPBS bottle.
Store at 4
C for up to 1 month.
5. 2 mM EDTA in DPBS supplemented with 2% FBS (PBMCbuffer): dissolve 58.4 mg of EDTA in 98 mL of DPBS, no
calcium, no magnesium. Filter-sterilize through a 0.22 μm
PTFE membrane. Add 2 mL of FBS and sonicate for 30 min.
Store at 4
C for up to 1 week.
6. Human Macrophage-Colony Stimulating Factor (hM-CSF)
stock (0.1 mg/mL): resuspend 100 μg of lyophilized
hM-CSF in 1 mL of sterile water. Store 100 μL aliquots at
À20
C. Avoid more than 3 freeze/thaw cycles.
7. CD14
+ beads: magnetic micro-beads conjugated to monoclonal anti-human CD14 antibodies (Miltenyi Biotec).
8. Magnet, column adaptors, and columns for magnetic separation (Miltenyi Biotec).
9. Sterile single-use beaker.
10. Sterile 144 cm
2 (120 Â 120 mm) culture petri dishes.
11. Laboratory centrifuge to achieve 330 Â g for 10 min and
850 Â g for 20 min (using a rotor with swing-out buckets).
2.11 Intracellular
Mycobacterial
Replication Assays
for siRNA Screening
N.B. Requirement of a sterile environment, such as a biosafety
cabinet in a dedicated laboratory.
1. RNase AWAY spray bottle (see Note 8).
2. Lipofectamine LTX reagent.
3. Lipofectamine RNAiMAX transfection reagent.
4. HiPerFect Transfection reagent.
5. RNase-free DEPC-treated water (see Note 8).
6. 5Â siRNA buffer (Horizon Discovery). Prepare 1Â siRNA
buffer by mixing 1 volume of 5Â siRNA buffer with 4 volumes
of RNase-free water.
7. siRNA solution: dilute 5 nmol lyophilized siRNA in 500 μL
DEPC-treated water to reach a final stock concentration of
10 μM. Vortex and keep on ice during 15 min before use.
Store stock solutions at À20
C.
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Nathalie Deboosere et al.
Cell Purification
and Culture
N.B. Requirement of a type 2 biosafety cabinet in a dedicated
biosafety laboratory adapted for pathogens of class 2.
1. See items 2–9 under Subheading 2.2.
2. Healthy volunteer peripheral blood samples.
3. Ficoll’s density gradient solution (density ¼ 1.077). Store at
room temperature in the dark.
4. DPBS, no calcium, no magnesium, supplemented with 1% FBS
(DPBS-FBS): add 5 mL of FBS in a 500 mL DPBS bottle.
Store at 4
C for up to 1 month.
5. 2 mM EDTA in DPBS supplemented with 2% FBS (PBMCbuffer): dissolve 58.4 mg of EDTA in 98 mL of DPBS, no
calcium, no magnesium. Filter-sterilize through a 0.22 μm
PTFE membrane. Add 2 mL of FBS and sonicate for 30 min.
Store at 4
C for up to 1 week.
6. Human Macrophage-Colony Stimulating Factor (hM-CSF)
stock (0.1 mg/mL): resuspend 100 μg of lyophilized
hM-CSF in 1 mL of sterile water. Store 100 μL aliquots at
À20
C. Avoid more than 3 freeze/thaw cycles.
7. CD14
+ beads: magnetic micro-beads conjugated to monoclonal anti-human CD14 antibodies (Miltenyi Biotec).
8. Magnet, column adaptors, and columns for magnetic separation (Miltenyi Biotec).
9. Sterile single-use beaker.
10. Sterile 144 cm
2 (120 Â 120 mm) culture petri dishes.
11. Laboratory centrifuge to achieve 330 Â g for 10 min and
850 Â g for 20 min (using a rotor with swing-out buckets).
2.11 Intracellular
Mycobacterial
Replication Assays
for siRNA Screening
N.B. Requirement of a sterile environment, such as a biosafety
cabinet in a dedicated laboratory.
1. RNase AWAY spray bottle (see Note 8).
2. Lipofectamine LTX reagent.
3. Lipofectamine RNAiMAX transfection reagent.
4. HiPerFect Transfection reagent.
5. RNase-free DEPC-treated water (see Note 8).
6. 5Â siRNA buffer (Horizon Discovery). Prepare 1Â siRNA
buffer by mixing 1 volume of 5Â siRNA buffer with 4 volumes
of RNase-free water.
7. siRNA solution: dilute 5 nmol lyophilized siRNA in 500 μL
DEPC-treated water to reach a final stock concentration of
10 μM. Vortex and keep on ice during 15 min before use.
Store stock solutions at À20
C.
656
Nathalie Deboosere et al.