following differences: (1) use D10 to resuspend scraped cells
and (2) J774 cells should be split 1:6 and passaged when
confluent, approximately every 3 days.
3.4 Maintenance and
Passage of THP-1 Cells
1. Thaw a vial of frozen THP cells in a 37
C water bath. Add the
entire volume within the vial to 10 mL of pre-warmed RPMI
complete medium in a 100 mm petri dish or T-25 tissue culture
flask.
2. Incubate overnight in a humidified incubator at 37
C and 5%
CO 2 .
3. Collect the cell suspensions in a conical tube and spin in a
swinging bucket, refrigerated centrifuge, at 200 Â g for
10 min at 4
C.
4. Discard supernatant and gently resuspend cell pellet in fresh,
pre-warmed RPMI complete medium to remove any residual
DMSO from the freezing media.
5. Incubate cells in a humidified incubator at 37
C and 5% CO 2
and check dishes or flasks daily for morphology, cell viability,
and cell number by observing any changes in media color and
counting cells with a hemacytometer.
6. Exchange the medium every 2–3 days.
7. THP-1 cells should be subcultured when a density of
8 Â 10
5 cells/mL is reached and not be allowed to exceed
1 Â 10
6 cells/mL. Passaged cells can be seeded at
2–4 Â 10
5 cells/mL.
8. To differentiate THP-1 cells into adherent cells with
macrophage-like properties, resuspend and seed cells
(5 Â 10
5 –1 Â 10
6 cells/mL) in the desired assay plate in
macrophage serum-free medium (Life Technologies, Grand
Island, NY) ammended with 100 nM phorbol 12-myristate
13-acetate (PMA) for 24 h. After 24 h, wash away nonadherent
cells with pre-warmed RPMI complete medium (5).
3.5 Maintenance,
Passage, and
Differentiation of
Conditionally
Immortalized
Estradiol-Inducible
HoxB8 Myeloid
Progenitor Cells
1. Thaw an entire vial of conditionally immortalized estradiolinducible HoxB8 cells (see Note 12 about generation of these
cells) in a 37
C water bath. Add the entire volume within the
vial to 10 mL of pre-warmed Hox medium in a sterile 15-mL
conical tube. Centrifuge the cells at 500 Â g for 5 min.
2. Discard the supernatant, resuspend the cells in 15 mL
pre-warmed Hox medium, and add the resuspended cells to a
100 mm tissue culture dish.
3. Undifferentiated HoxB8 cells grow in suspension in tissue
culture dishes. Media should be replaced every 3 days with
fresh Hox medium to ensure that the estradiol concentration
remains optimal. Cells should not exceed a concentration of
~500,000 cells per mL prior to differentiation.
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Benjamin K. Johnson et al.
and (2) J774 cells should be split 1:6 and passaged when
confluent, approximately every 3 days.
3.4 Maintenance and
Passage of THP-1 Cells
1. Thaw a vial of frozen THP cells in a 37
C water bath. Add the
entire volume within the vial to 10 mL of pre-warmed RPMI
complete medium in a 100 mm petri dish or T-25 tissue culture
flask.
2. Incubate overnight in a humidified incubator at 37
C and 5%
CO 2 .
3. Collect the cell suspensions in a conical tube and spin in a
swinging bucket, refrigerated centrifuge, at 200 Â g for
10 min at 4
C.
4. Discard supernatant and gently resuspend cell pellet in fresh,
pre-warmed RPMI complete medium to remove any residual
DMSO from the freezing media.
5. Incubate cells in a humidified incubator at 37
C and 5% CO 2
and check dishes or flasks daily for morphology, cell viability,
and cell number by observing any changes in media color and
counting cells with a hemacytometer.
6. Exchange the medium every 2–3 days.
7. THP-1 cells should be subcultured when a density of
8 Â 10
5 cells/mL is reached and not be allowed to exceed
1 Â 10
6 cells/mL. Passaged cells can be seeded at
2–4 Â 10
5 cells/mL.
8. To differentiate THP-1 cells into adherent cells with
macrophage-like properties, resuspend and seed cells
(5 Â 10
5 –1 Â 10
6 cells/mL) in the desired assay plate in
macrophage serum-free medium (Life Technologies, Grand
Island, NY) ammended with 100 nM phorbol 12-myristate
13-acetate (PMA) for 24 h. After 24 h, wash away nonadherent
cells with pre-warmed RPMI complete medium (5).
3.5 Maintenance,
Passage, and
Differentiation of
Conditionally
Immortalized
Estradiol-Inducible
HoxB8 Myeloid
Progenitor Cells
1. Thaw an entire vial of conditionally immortalized estradiolinducible HoxB8 cells (see Note 12 about generation of these
cells) in a 37
C water bath. Add the entire volume within the
vial to 10 mL of pre-warmed Hox medium in a sterile 15-mL
conical tube. Centrifuge the cells at 500 Â g for 5 min.
2. Discard the supernatant, resuspend the cells in 15 mL
pre-warmed Hox medium, and add the resuspended cells to a
100 mm tissue culture dish.
3. Undifferentiated HoxB8 cells grow in suspension in tissue
culture dishes. Media should be replaced every 3 days with
fresh Hox medium to ensure that the estradiol concentration
remains optimal. Cells should not exceed a concentration of
~500,000 cells per mL prior to differentiation.
174
Benjamin K. Johnson et al.