Culture, Isolation, and Labeling of Anaplasma phagocytophilum for Subsequent Infection of Human Neutrophils - Bacterial Pathogenesis: Methods and Protocols - page 151

Successful culture of A. phagocytophilum was first described in 1996 (3) .
Since that time, pathogen culture, isolation, and labeling techniques have rapidly
been developed to facilitate the study of pathogen-host cell interactions. The
techniques described in this chapter highlight standard and pathogen-specific
culture and isolation techniques. Options for labeling A. phagocytophilum for
use in flow cytometric and immunofluorescence protocols are described with
an emphasis on a labeling technique that facilitates the distinction between
intra- and extracellular organisms. This technique permits ready dissection of
pathogen binding, entry, and infection of human neutrophils. In addition, the
techniques described could be readily modified for study of pathogen interaction
with a broad array of host cells.

2. Materials
2.1. Anaplasma phagocytophilum Culture and Propagation
in HL60 Cells
1. Human myeloid leukemia cell line (HL60 cells, ATCC 240-CCL; American
Type Culture Collection, Rockville, MD). Upon receipt, HL60 cells should be
stored in liquid nitrogen ( see Note 1 ).
2. RPMI-1640 media supplemented with 10% heat-inactivated fetal bovine serum
(FBS) and 2 m M l-glutamine ( see Note 2 ).
3. Nunc cryovials.
4. Standard cell-culture flasks (25 cm 2 and 75 cm 2 flasks, canted-neck polystyrene,
non-pyrogenic; BD Biosciences, San Jose, CA, USA).
5. 0.2 μm vented blue plug seal cap ( see Note 3 ).
6. Sterile 1-25-mL pipettes.
7. 15- and 50-mL polystyrene conical tubes.
8. 500-mL top filter with 45-mm neck.
9. 0.22-μm cellulose acetate, sterilizing,

low protein binding membrane non-

pyrogenic (Corning, NY, USA).
10. Freezing media: 90% cell-culture media (RPMI-1640 media containing 10%
FBS) and 10% dimethysulfoxide (DMSO).
11. Infected, EDTA-anticoagulated patient blood. Sources of infected blood may
include infected human beings, mice, horses, dogs, or ruminants. Blood must be
sterile.
12. All- trans -retinoic acid (1 μ M retinoic acid made froma1m M stock solution
prepared in 95% (v/v) ethanol and stored at -80 degrees; Sigma) or 1.25%
DMSO (Sigma-Aldrich, St.Louis, MO, USA).
13. 1,25-dihydroxyvitamin D 3 (2 μ M Vitamin D 3 ; Calbiochem, San Diego, CA)
prepared as a 10 4 M stock solution by dissolving in 95% ethanol and stored
at -20 °C.