into a conical tube with 5 mL of D10 medium and centrifuge at
300 Â g for 5 min. Remove the supernatant and resuspend in
7 mL of D10 medium and transfer to a T-25 tissue culture
flask. Incubate at 37
C and 5% CO 2 until cells are confluent,
about 4–5 days. Once confluent, remove the supernant, wash
once with sterile PBS, and then add 5 mL of PBS with 0.25%
trypsin (w/v) and 0.53 mM EDTA and incubate for 5–10 min
until the cells detach. Centrifuge cells at 300 Â g for 5 min and
resuspend in 10 mL of D10. Use this culture to seed 1 mL of
culture into 30 mL of D10 into a T-150 flask and incubate flask
at 37
C and 5% CO 2 until cells are 90% confluent (approximately 4–5 days). Passage cells as described above (but using
10 mL of PBS–Trypsin–EDTA to detach cells) and resuspend
in 4 mL of D10. Use 1 mL of cells to seed a triple layer flask
(e.g., Nunc Triple flask 500 cm
2 with vent cap) containing
180 mL of modified D10 medium with reduced antibiotics
(a final concentration of 10 I.U./mL of penicillin and 10 μg/
mL of streptomycin). Incubate the triple-layer flasks at 37
C
and 5% CO 2 for approximately 1 week, mixing the flasks every
other day. To harvest L-cell conditioned medium, pour supernatants into sterile endotoxin-free tubes (e.g., 50 mL conical
tubes) and centrifuge at 1800 Â g for 10 min. Pour the supernatants into the fresh 50 mL conical tubes and freeze at
À20
C. The highest level of macrophage differentiating cytokines will be obtained shortly before the cells begin to die and
the medium turns orange. Harvest the supernatant as soon as
the color of the medium begins to change.
3. To prepare the OADC supplement, dissolve 8.5 g NaCl and
50 g of BSA fraction V in 900 mL of ddH 2 O. We have found it
useful to gently heat and stir the solution as the BSA tends to
clump and go into solution slowly. Dissolve 20 g D-dextrose,
40 mg catalase, and 0.5 g oleic acid into the solution. Bring the
final volume up to 1 L. Aliquot and centrifuge the solution in
500 mL bottles for 30 min at 1800 Â g, 4
C. Sterilize supernatants with a 0.22 μm filter sterilization unit and aliquot into
50 mL conical tubes. Store at 4
C. The OADC supplement is
also available for purchase (Sigma-Aldrich Corp.,
St. Louis, MO).
4. Do not add OADC or cycloheximide to 7H10 agar medium
before autoclaving. Warm the OADC and cyclohexamide to
55
C and add to the 7H10 agar cooled to 55
C. Mix thoroughly and dispense agar into quadrant plates. To dry plates,
cover and stack plates and leave in a closed biological safety
cabinet for 2 days.
5. Hazardous: Prepare 4% PFA in a chemical fume hood. Heat a
glass beaker of stirred PBS on a hot plate to approximately
60
C. Add PFA to heated PBS; however, it will not dissolve
180
Benjamin K. Johnson et al.
300 Â g for 5 min. Remove the supernatant and resuspend in
7 mL of D10 medium and transfer to a T-25 tissue culture
flask. Incubate at 37
C and 5% CO 2 until cells are confluent,
about 4–5 days. Once confluent, remove the supernant, wash
once with sterile PBS, and then add 5 mL of PBS with 0.25%
trypsin (w/v) and 0.53 mM EDTA and incubate for 5–10 min
until the cells detach. Centrifuge cells at 300 Â g for 5 min and
resuspend in 10 mL of D10. Use this culture to seed 1 mL of
culture into 30 mL of D10 into a T-150 flask and incubate flask
at 37
C and 5% CO 2 until cells are 90% confluent (approximately 4–5 days). Passage cells as described above (but using
10 mL of PBS–Trypsin–EDTA to detach cells) and resuspend
in 4 mL of D10. Use 1 mL of cells to seed a triple layer flask
(e.g., Nunc Triple flask 500 cm
2 with vent cap) containing
180 mL of modified D10 medium with reduced antibiotics
(a final concentration of 10 I.U./mL of penicillin and 10 μg/
mL of streptomycin). Incubate the triple-layer flasks at 37
C
and 5% CO 2 for approximately 1 week, mixing the flasks every
other day. To harvest L-cell conditioned medium, pour supernatants into sterile endotoxin-free tubes (e.g., 50 mL conical
tubes) and centrifuge at 1800 Â g for 10 min. Pour the supernatants into the fresh 50 mL conical tubes and freeze at
À20
C. The highest level of macrophage differentiating cytokines will be obtained shortly before the cells begin to die and
the medium turns orange. Harvest the supernatant as soon as
the color of the medium begins to change.
3. To prepare the OADC supplement, dissolve 8.5 g NaCl and
50 g of BSA fraction V in 900 mL of ddH 2 O. We have found it
useful to gently heat and stir the solution as the BSA tends to
clump and go into solution slowly. Dissolve 20 g D-dextrose,
40 mg catalase, and 0.5 g oleic acid into the solution. Bring the
final volume up to 1 L. Aliquot and centrifuge the solution in
500 mL bottles for 30 min at 1800 Â g, 4
C. Sterilize supernatants with a 0.22 μm filter sterilization unit and aliquot into
50 mL conical tubes. Store at 4
C. The OADC supplement is
also available for purchase (Sigma-Aldrich Corp.,
St. Louis, MO).
4. Do not add OADC or cycloheximide to 7H10 agar medium
before autoclaving. Warm the OADC and cyclohexamide to
55
C and add to the 7H10 agar cooled to 55
C. Mix thoroughly and dispense agar into quadrant plates. To dry plates,
cover and stack plates and leave in a closed biological safety
cabinet for 2 days.
5. Hazardous: Prepare 4% PFA in a chemical fume hood. Heat a
glass beaker of stirred PBS on a hot plate to approximately
60
C. Add PFA to heated PBS; however, it will not dissolve
180
Benjamin K. Johnson et al.