Culture, Isolation, and Labeling of Anaplasma phagocytophilum for Subsequent Infection of Human Neutrophils - Bacterial Pathogenesis: Methods and Protocols - page 152

14. 12- O -tetradecanoylphorbol-13-acetate (TPA; Sigma) prepared as a 1.6 × 10 − 4
M stock in DMSO and stored at -80 °C for ≤ 6 months.

2.2. Isolation of Cell-Free Anaplasma phagocytophilum
1. Hematocytometer, 1.5-mL microfuge tubes, 1-500-μL pipettor, trypan blue,
microscope slides, modified Romanosky stain set (i.e., Hemaquick), and glass
microfiber filters (25-nm GD/X filter).
2. 3-9-mL sterile syringes and 25-27.5 gauge needles, or sonic dismembranator.
3. Cytofuge (Shandon Cytospin; ThermoShandon, Pittsburgh, PA, USA).
4. SPGN buffer: 7.5% sucrose, 3.7 m M KH 2 PO 4 ,7m M K 2 HPO 4 ,and5m M
glutamine, pH 7.4) ( see Note 4 ).
5. Diatrizoate meglumine (66% diatrizoate meglumine and 10% diatrizoate sodium
in 0.32% sodium citrate and 0.04% edentate disodium) should be obtained as
a 76% solution. Diatrizoate meglumine is an ionic radiopaque contrast agent.
Brand names include Renografin 76, Hypaque 76, and Angiovist 370. Dilutions
of diatrizoate meglumine can be made in PBS buffer.

2.3. Determining Bacterial Infectious Dose
1. Hematocytometer, microscope slides, and modified Romanosky stain set (i.e.,
Hemaquick).

2.4. Bacteria Labeling and Modifications
1. Specific purified monoclonal antibodies that label outer membrane proteins of
A. phagocytophilum may be obtained from non-commercial sources (e.g., R1B10,
R5E4, and R5A9) (4) .
2. Zenon Mouse IgG Labeling Kit (Invitrogen Corp., Carlsbad, CA, USA) to label
unconjugated antibodies.
3. Serum-free protein block (DAKO, Carpinteira, CA, USA).
4. Methanol, PBS, coverslips, and bovine serum albumin (BSA).
5. Fluorescent dyes including the lipophilic fluorescent compound PKH-67 (Sigma)
and Cell Tracker Green (CMFDA; Invitrogen) can be used to label A. phagocy-
tophilum for immunofluorescent assays or flow cytometry (5) .
6. Doxycycline (10 μg/mL; Sigma) or oxytetracycline hydrochloride (10 mg/mL;
Sigma).
7. Paraformaldehyde (2% solution dissolved in PBS).

2.5. Incubation of Bacteria with Neutrophils
1. RPMI-1640 media buffered with 10 m M HEPES, pH 7.4.
2. 24-well tissue-culture plates.
3. AlexaFluor 488, anti-AlexaFlour 488 conjugated to AlexaFlour 594 (Invitrogen,
Carlsbad, CA, USA).