replicative plasmid (based on pMV261) carrying a pDSRed1–1
(Clontech) gene under the Hsp60 promoter and conferring
resistance to kanamycin. M. tuberculosis Beijing GC1237mCherry are recombinant strains bearing the pCherry10 plasmid [21], conferring resistance to hygromycin B.
2. Routinely, we used either PerkinElmer CellCarrier-384 (Optically Clear Bottom, tissue culture treated, black, sterile with lid)
or Greiner Bio-one, Cat 781091 (384-well polystyrene cell
culture microplates, Flat Bottom, μClear, black, sterile
with lid).
3. Cells can be stored in liquid nitrogen for long-term storage.
Typically, 1 mL aliquots containing 5 Â 10
6 RAW264.7,
THP-1, A549, L929, or J558 cells in FBS containing 10%
DMSO are prepared during cell isolation. After thawing the
aliquot and tenfold dilution into pre-warmed (37
C) RPMIFBS, cells are pelleted by centrifugation for 5 min at 240 Â g at
room temperature to remove DMSO and resuspended in fresh,
pre-warmed RPMI-FBS.
4. Fetal bovine serum (FBS) can interfere with the cell phenotypes. Perform infection assays with your new FBS stock in
comparison with the previous one to compare the bacterial
growth.
5. Macrophage and dendritic progenitor cells can be stored in
liquid nitrogen for longer term storage.
6. Before using a new batch of growth factor-enriched supernatant, the percentage of L929-derived M-CSF conditioned
medium needs to be determined to obtain good differentiation
of isolated monocytes into macrophages. We test different
percentages of M-CSF on monocytes and follow the described
protocol for murine primary macrophage culture (see Subheading 3.8). Cell suspensions are then incubated with either specific antibodies of macrophage surface markers (detection of
F4/80, CD11b, and MHC-I) and specific antibodies to cell
surface markers, which should be absent in BMDM (e.g.,
CD11c). We select the percentage, which shows the best differentiation rate using fluorescence-activated cell sorting
(FACS).
7. Before using a new batch of growth factor-enriched supernatant, the percentage of J558-derived GM-CSF conditioned
medium needs to be determined to obtain good differentiation
of hematopoietic stem cells into dendritic cells. We usually test
different percentages of GM-CSF on hematopoietic stem cells
and follow the described protocol for murine primary dendritic
cell culture (see Subheading 3.9). Cell suspensions are then
incubated with either specific antibodies of dendritic cell surface markers (detection of F4/80, CD11c, and MHC-II) and
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Nathalie Deboosere et al.
(Clontech) gene under the Hsp60 promoter and conferring
resistance to kanamycin. M. tuberculosis Beijing GC1237mCherry are recombinant strains bearing the pCherry10 plasmid [21], conferring resistance to hygromycin B.
2. Routinely, we used either PerkinElmer CellCarrier-384 (Optically Clear Bottom, tissue culture treated, black, sterile with lid)
or Greiner Bio-one, Cat 781091 (384-well polystyrene cell
culture microplates, Flat Bottom, μClear, black, sterile
with lid).
3. Cells can be stored in liquid nitrogen for long-term storage.
Typically, 1 mL aliquots containing 5 Â 10
6 RAW264.7,
THP-1, A549, L929, or J558 cells in FBS containing 10%
DMSO are prepared during cell isolation. After thawing the
aliquot and tenfold dilution into pre-warmed (37
C) RPMIFBS, cells are pelleted by centrifugation for 5 min at 240 Â g at
room temperature to remove DMSO and resuspended in fresh,
pre-warmed RPMI-FBS.
4. Fetal bovine serum (FBS) can interfere with the cell phenotypes. Perform infection assays with your new FBS stock in
comparison with the previous one to compare the bacterial
growth.
5. Macrophage and dendritic progenitor cells can be stored in
liquid nitrogen for longer term storage.
6. Before using a new batch of growth factor-enriched supernatant, the percentage of L929-derived M-CSF conditioned
medium needs to be determined to obtain good differentiation
of isolated monocytes into macrophages. We test different
percentages of M-CSF on monocytes and follow the described
protocol for murine primary macrophage culture (see Subheading 3.8). Cell suspensions are then incubated with either specific antibodies of macrophage surface markers (detection of
F4/80, CD11b, and MHC-I) and specific antibodies to cell
surface markers, which should be absent in BMDM (e.g.,
CD11c). We select the percentage, which shows the best differentiation rate using fluorescence-activated cell sorting
(FACS).
7. Before using a new batch of growth factor-enriched supernatant, the percentage of J558-derived GM-CSF conditioned
medium needs to be determined to obtain good differentiation
of hematopoietic stem cells into dendritic cells. We usually test
different percentages of GM-CSF on hematopoietic stem cells
and follow the described protocol for murine primary dendritic
cell culture (see Subheading 3.9). Cell suspensions are then
incubated with either specific antibodies of dendritic cell surface markers (detection of F4/80, CD11c, and MHC-II) and
698
Nathalie Deboosere et al.