Culture, Isolation, and Labeling of Anaplasma phagocytophilum for Subsequent Infection of Human Neutrophils - Bacterial Pathogenesis: Methods and Protocols - page 154

Fig. 1. Images of Anaplasma phagocytophilum -infected HL60 cells. (A) Cytofuge
smear of highly infected HL60 cells. Arrow indicates intact, multiple pathogen
morulae within the HL60 cell cytoplasm (6) . Arrowhead indicates HL60 cell lysis
with abundant extracellular bacteria (1000× magnification; Hema-Quik stain). (B)
Electron micrograph of a highly infected HL60 cell with abundant intracellular
bacteria found within cytoplasmic vacuoles (morula). The N labels the cell nucleus.
Arrows indicate the “reticulate,” possibly replicative, stage of organism development.
The arrowhead indicates the “dense cored,” possibly infective, stage of organism
development. ( See Color Plate 5, following p. 46.)
3.1.1. Initiation of Infected HL60 Cells Through Cultivation of Patient
Blood Samples in HL60 Cells
1. Sterile-inoculate 100 μL of patient EDTA-anticoagulated whole blood into 5 mL
of5×10 5 cells/mL HL60 cells in a 25-cm 2 cell-culture flask. Infection will
typically be noted by day 5 post-inoculation; however, up to 15 days can be
needed for cultures to be 90% infected. Variation in infectivity is noted with some
patient samples resulting in <5% infection even after 2 weeks. Blood samples
may be stored at 4 °C for up to 48 h before inoculation into culture. Diagnostic
culture of organisms from patient blood may be enhanced by addition of retinoic
acid to HL60 cells ( see Subheading 2.1.2. ). Cultures may be negative after only
24 h of appropriate antibiotic therapy.
3.1.2. Induction of Granulocytic or Monocytic Differentiation
1. Incubate5×10 5 -1×10 6 cells/mL HL60 cells for 3-6 days with 1 μ M all- trans -
retinoic acid (7) or 1.25% DMSO (8) . After 6 days of exposure, approximately
half of the cells will appear granulocytic and show myeloperoxidase staining ( see
Note 7 ).
2. Incubate5×10 5 -1×10 6 cells/mL HL60 for 3-6 days with 2 μ M 25-OH vitamin
D 3 or with the protein kinase C activator, TPA, at 1.6 × 10 − 7 -10 − 10 M final
concentration to induce macrophage differentiation (8) ( see Note 7 ).