Culture, Isolation, and Labeling of Anaplasma phagocytophilum for Subsequent Infection of Human Neutrophils - Bacterial Pathogenesis: Methods and Protocols - page 155

3.2. Isolation of Cell-Free Anaplasma phagocytophilum
3.2.1. Routine Infection Studies
1. Count infected HL60 cells by removing 150 μL of cells from culture flask.

Dilute 10 μL of well-mixed cells into 90 μL of trypan blue and count cells using
a hematocytometer. The use of trypan blue permits concurrent assessment of cell
viability.
2. Estimate percent infection. Take remaining undiluted cells and make a cytofuge
smear (use a Shandon Cytospin at 70 × g for 5 min). Air-dry the smear, fix with
methanol and stain with any modified Romanosky stain (e.g., Hema-Quik, Dif-
Quick, and Wright-Giemsa). With a light microscope, determine the percent of
infected HL60 cells by counting 100 cells ( see Fig. 1A ). Alternatively, if a cytospin
is unavailable, centrifuge

∼

300 μL of infected HL60 cells at 450 × g for 5 min
to sediment cells. Remove supernatant and resuspend cells in ∼ 50 μL of saline or
PBS. Place a large drop of sedimented cells unto a microscope slide and smear
the cells in a fashion similar to that of a blood smear. Stain and count as described.
3. Disrupt HL60 cells for pathogen isolation by cell sonication or needle lysis. To
sonicate, resuspend desired number of cells in

∼

5 mL of media and place in
a 50-mL conical tube. Place tubes on ice. Sonicate cells gently with 3-5 quick
pulses. Alternatively, cells can be lysed by 3-6 passages through a 25-27.5 gauge
needle ( see Note 8 ).
4. Centrifuge sonicate or lysate to pellet unwanted cellular debris (450 × g for 5 min;
A. phagocytophilum is in supernatant). Filter supernatant with glass microfiber
filter to remove remnant HL60 debris and further clarify the lysate. Pellet the
pathogen by centrifugation of supernatant (5000 × g for 10-15 min). Discard
supernatant and resuspend pelleted A. phagocytophilum in working media/buffer.
Wash one time by pelleting pathogen as above (5000 × g for 10-15 min).

∼

3.2.2. Purification Process
To obtain a more pure population of A. phagocytophilum , organisms can be
isolated from host cell debris using a number of techniques ( see Note 9 ). The
most common protocol uses diatrizoate meglumine. This technique was first
described for the Rickettsiae in 1981 and has been modified over time by a
number of investigators (9,10,11,12) ( see Note 10 ).
1. Follow steps 1-3 in Subheading 3.2.1 . High numbers of infected HL60 cells
(i.e., 2-6 × 10 8 cells) are needed for this process.
2. Centrifuge sonicate or lysate to pellet unwanted cellular debris (450 × g for 5-10
min; A. phagocytophilum is in supernatant). Pellet pathogen by centrifugation of
supernatant (5000 × g for 10-15 min). Discard supernatant and resuspend pelleted
A. phagocytophilum in SPGN buffer.
3. Layer resuspend A. phagocytophilum onto a discontinuous gradient of 6-8 mL of
42% and 10-12 mL of 30% diatrizoate meglumine ( see Note 11 ). Ultracentrifuge
gradients to equilibrium at 50,000 × g for 60-75 min at 4 °C. Bacterial fractions