Culture, Isolation, and Labeling of Anaplasma phagocytophilum for Subsequent Infection of Human Neutrophils - Bacterial Pathogenesis: Methods and Protocols - page 159

2. Anaplasma phagocytophilum grown in HL60 cells may also be cultured in
Iscove's modified Dulbecco's medium (IMDM) or Dulbecco's modified Eagle's
medium (DMEM) supplemented with 20% FBS. Differences in pathogen
kinetics and growth in these different media have not been appreciated or
reported. Different investigators have varied the percent of FBS in the media
from 1-20%.
3. Top vented cell-culture flasks are highly recommended, as antibiotics are not
used in cell-culture systems for A. phagocytophilum . The use of these flasks
decreases the risk of secondary bacterial contamination.
4. Monocytic Ehrlichia metabolize best in alkaline pH (7.2-8.0; adjust with 2 M
NaOH), the use of slightly alkaline pH has been maintained for the related
A. phagocytophilum . Sucrose phosphate buffer provides osmotic protection for
these fragile organisms. Membrane active agents (phospholipase, detergent) and
low-osmolarity buffers or osmotic shock are all deleterious to Ehrlichial viability
(10) . Glutamine is the preferred metabolic substrate for organisms (18) .
5. Keep track of pathogen passage number. Organisms passaged a high number
of times may have different virulence characteristics than low-passaged
pathogens. In addition, highly passaged HL60 cells shed P-selectin glyco-
protein ligand-1 (PSGL-1). PSGL-1 serves as a pathogen receptor (5) . If poor
growth or propagation of organisms is noted (suspect this when cells fail to
increase infectivity >50-60% in spite of high numbers of intact, uninfected
HL60 cells), assess PSGL-1 expression on the surface of feeder uninfected
HL60 cells.
6. Harvest pathogen when HL60 cells are at peak infection, when both intracellular
and extracellular organisms are noted ( see Fig. 1A ). Ultrastructural evaluation
of A. phagocytophilum reveals both reticulate and dense morphologic forms,
the biologic significance of which is unknown ( see Fig. 1B ) (19) . Recent work
with the related E. chaffeensis suggests that the dense-cored form may be the
infective stage and the reticulate form may be the replicative stage ( see Fig. 1B )
(20) . Empirically, pathogen isolated from HL60 cell cultures that are only

≤ 50%

infected (early in infection) may function differently in assay systems.
7. Pathogen growth can be sustained in both granulocyte-induced and uninduced
HL60 cell cultures; however, growth of A. phagocytophilum will not be sustained
in monocyte-differentiated HL60 cells (8,21) . Preferential growth of organism
occurs in HL60 cells differentiated toward granulocytes, especially when differ-
entiated with DMSO. DMSO differentiation results in heterogeneity of CD15
surface expression. However, cells with high levels of expression show greatest
binding and infection of organism (21) .
8. Sonication conditions will vary depending on sonicator used. Cell lysis is readily
evident when foaming of media occurs. The organism is fragile, and thus minimal
and gentle sonication is recommended. Empirically, we have lysed cells for