Culture, Isolation, and Labeling of Anaplasma phagocytophilum for Subsequent Infection of Human Neutrophils - Bacterial Pathogenesis: Methods and Protocols - page 157

3.4.2. Labeling with Fluorescent Dyes
1. Dilute stock CMFDA (CellTracker Green at 10 m M )to15μ M final concentration
in pre-warmed media with no FBS.
2. Add 500 μL of 15 μ M CMFDA to a 500 μL aliquot of bacteria (isolated as
described in Subheading 3.2.1. ).
3. Mix well. Incubate at 37 °C for 45 min.
4. Centrifuge at 5000 × g for 10 min to pellet bacteria.
5. Wash by resuspending in 500 μL of prewarmed media and centrifuging at 5000 × g
for 10 min to pellet bacteria.
6. Resupend in 500 μL of working media ( see Note 12 ).
3.4.3. Treatments Before Incubation with Neutrophils
Anaplasma phagocytophilum can be modified to assess the role of viability
in measured outcomes.
1. To heat kill A. phagocytophilum , take isolated pathogen suspended in working
buffer and heat in a water bath or heating block at 60-100 °C for 10 min (13) .
2. Exposure to doxycycline or oxytetracycline hydrochloride for 30 min at room
temperature also inactivates A. phagocytophilum and renders the organism non-
infective for HL60 cell culture.
3. To fix A. phagocytophilum , suspend isolated pathogen in 2% paraformaldehyde
in working buffer for 30 min at room temperature.
3.5. Incubation of Bacteria with Neutrophils
1. Isolate human neutrophils according to standard protocols (13) . Resuspend
neutrophils in RPMI/HEPES to 2 × 10 6 cells/mL. Keep cells at room temperature
until they are added to the tissue-culture plate ( see Note 13 ).
2. Resuspend isolated A. phagocytophilum in 1 mL PBS containing 7.5 μg/mL
Alexa Fluor 488 (Invitrogen-Molecular Probes). Incubate for 15 min at room
temperature. Wash twice in 1 mL PBS to remove unbound AlexaFluor 488.
Resuspend bacteria in RPMI/HEPES at desired concentration to add a volume
of 250 μL of suspended bacteria to each well.
3. Immerse #1, 12-mm round glass coverslips in ethanol and flame. Place one
clean coverslip in each well of a 24-well tissue-culture plate and let sit under
UV light in a tissue-culture hood.
4. Coat each coverslip with 30 μL of 100% autologous human serum. Incubate for
at least1hat37°C.Wash twice with PBS.
5. Add 250 μL of human neutrophils to each well and let cells settle and adhere for
15 min at room temperature. Chill culture plates with neutrophils on ice until
addition of bacteria.
6. Add 250 μL of AlexaFluor 488-labeled A. phagocytophilum to each well and
centrifuge at 900 × g for 8 min at 4 °C to synchronize phagocytosis. Transfer
all plates except 0 min time point to 37 °C.