into solution. Add 1 N NaOH until the PFA goes into solution. Allow solution to cool, bring it up to volume with PBS,
and filter. Check the pH of the PFA and if necessary adjust it to
pH 7.4 (using pH strips instead of pH meter). Freeze aliquots
of PFA for single use. Do not freeze and thaw PFA multiple
times.
6. We have found it useful to use a small pair of pliers to bend the
end of a syringe needle (23g1) into a small hook. The hook can
be used to gently break the surface tension between the glass
slide and the plastic bottom of the well. This step requires
special care to avoid breaking coverslips.
7. It is essential that one is in compliance with the specific Institutional Animal Care and Use Committee (IACUC) approved
methods for the care and euthanasia of mice.
8. Be careful to not incubate the cells at 4
C for more than
10 min because cell viability may decrease.
9. It is particularly important to scrape along the edges because
cells tend to accumulate at the edges of the dish.
10. When counting cells, observe and count four, 1 mm  1 mm
squares (e.g., the four corners of the hemacytometer). Take the
average number of cells and multiply by 10
4 to obtain cells/
mL.
11. When seeding 24-well, 96-, or 384-well plates, addition of the
following amounts of cells will result in confluent wells within
24 h. For 24-well plates, add 0.5 mL of cells at
1–1.5 Â 10
6 cells/mL to each well; for a 96-well plate, add
80 μL of cells at approximately 2 Â 10
6 cells/mL per well; and
for a 384-well plate, add 20 μL of of cells at approximately
2 Â 10
6 cells/mL per well. When preparing 384-well plates,
after the addition of macrophages, centrifuge the assay plates at
100 Â g for 5 min to ensure cells are in contact with the bottom
of the well.
12. To generate conditionally immortalized myeloid progenitors
using estradiol-inducible HoxB8 conditionally immortalized
macrophages, follow the detailed protocol from Wang
et al. [6].
13. Working with sharps in the BSL-3 represents a potential safety
hazard and requires training prior to use. Blunt needle syringes
are available and can be substituted for safety purposes.
14. For infection of J774 or THP-1 cells, perform experiments as
described for primary murine bone marrow-derived macrophages, but subsitute macrophage infection medium with
D10 medium or RPMI complete medium, respectively, and
omit the antibiotics from both media.
Macrophage Infection Models for Mycobacterium tuberculosis
181
and filter. Check the pH of the PFA and if necessary adjust it to
pH 7.4 (using pH strips instead of pH meter). Freeze aliquots
of PFA for single use. Do not freeze and thaw PFA multiple
times.
6. We have found it useful to use a small pair of pliers to bend the
end of a syringe needle (23g1) into a small hook. The hook can
be used to gently break the surface tension between the glass
slide and the plastic bottom of the well. This step requires
special care to avoid breaking coverslips.
7. It is essential that one is in compliance with the specific Institutional Animal Care and Use Committee (IACUC) approved
methods for the care and euthanasia of mice.
8. Be careful to not incubate the cells at 4
C for more than
10 min because cell viability may decrease.
9. It is particularly important to scrape along the edges because
cells tend to accumulate at the edges of the dish.
10. When counting cells, observe and count four, 1 mm  1 mm
squares (e.g., the four corners of the hemacytometer). Take the
average number of cells and multiply by 10
4 to obtain cells/
mL.
11. When seeding 24-well, 96-, or 384-well plates, addition of the
following amounts of cells will result in confluent wells within
24 h. For 24-well plates, add 0.5 mL of cells at
1–1.5 Â 10
6 cells/mL to each well; for a 96-well plate, add
80 μL of cells at approximately 2 Â 10
6 cells/mL per well; and
for a 384-well plate, add 20 μL of of cells at approximately
2 Â 10
6 cells/mL per well. When preparing 384-well plates,
after the addition of macrophages, centrifuge the assay plates at
100 Â g for 5 min to ensure cells are in contact with the bottom
of the well.
12. To generate conditionally immortalized myeloid progenitors
using estradiol-inducible HoxB8 conditionally immortalized
macrophages, follow the detailed protocol from Wang
et al. [6].
13. Working with sharps in the BSL-3 represents a potential safety
hazard and requires training prior to use. Blunt needle syringes
are available and can be substituted for safety purposes.
14. For infection of J774 or THP-1 cells, perform experiments as
described for primary murine bone marrow-derived macrophages, but subsitute macrophage infection medium with
D10 medium or RPMI complete medium, respectively, and
omit the antibiotics from both media.
Macrophage Infection Models for Mycobacterium tuberculosis
181