Biology Reference
In-Depth Information
4. No. 1, 12-mm round glass coverslips.
5. Microscope slides, mounting media.
3. Methods
3.1. Anaplasma phagocytophilum Culture and Propagation
in HL60 Cells
1. Prepare1LofRPMI-1640 containing 10% FBS cell-culture media (RPMI/10%
FBS) by placing 100 mL of heat-inactivated FBS into 900 mL RPMI-1640 media.
Filter-sterilize using a 0.22-μm pore size bottle top filter. Store at 4 °C for up to
6 months.
2. To initiate HL60 culture, pipette 9 mL of RPMI/10% FBS media into a 15-mL
conical tube. Thaw one vial of HL60 cells in a 37 °C water bath for 2-3 min. Pipette
thawed cells into the 9 mL of media. Spin at 450 ×
g
for 5 min to pellet cells.
Pour off freezing media supernatant. Resuspend cells in 5 mL of fresh media and
place in 25-cm
2
flask. Cells should be maintained at 37 °C and 5% CO
2
/ 95% air.
3. The initiation of infected HL60 cell culture is identical to that described above
(#2); however, 5 mL of uninfected HL60 cells in fresh media should be added
to the 1 mL of 90% thawed/infected cells to a final concentration of
∼
5×10
5
cells/mL in a 25-cm
2
flask.
4. To maintain uninfected cells in culture, keep cell density from 5 × 10
5
to2×10
6
cells/mL by feeding the cells with fresh media twice a week. Uninfected cells grow
slower than infected cells, so uninfected cells should be thawed and propagation
initiated before infected cells. In addition, maintain at least one additional flask
of uninfected HL60 cells to feed infected cell cultures. Uninfected cells can be
expanded to a 75-cm
2
flask by taking 10 mL of uninfected cells and adding 10
mL of fresh media.
5. To maintain infected cells in culture, check infection level every 2-3 days (see
Section 3.2.1). Initial infection is often noted by day 3 with peak infection by
day 5-10. Culture will be > 90% infected by day 5 (
see
Note 5; Fig. 1A
and
Color Plate 5, following p. 46). Cell lysis will be noted by day 12-14. Add 1:1
fresh uninfected to infected HL60 cells twice weekly.
A. phagocytophilum
has a
cytotoxic effect on HL60 cells and will induce cell lysis and apoptosis if they
become too infected
(6)
. Infected cells can also be expanded to a 75-cm
2
flask by
adding 2 mL of highly infected HL60 cells diluted with 8 mL uninfected HL60
cells and 10-15 mL fresh media. Harvest pathogen from infected cell cultures at
peak infection (>90% infected HL60 cells (
see
Note 6
).
6. To freeze uninfected and infected HL60 cells, transfer media and cells from
tissue-culture flask into 50 mL conical tubes. Centrifuge at 450 ×
g
for 5 min
to pellet cells. Decant or aspirate supernatant and discard. Resuspend 1 mL of
cells at a final concentration of 5 × 10
6
cells/mL in freezing medium. Transfer
contents (
0.5 mL per tube) into NUNC cryovials. Freeze at -80 °C overnight,
then transfer vials to liquid nitrogen the next day. Freeze infected HL60 cells
at
∼
∼
90% infection level.
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