Biology Reference
In-Depth Information
can be collected with a sterile pipette or through punctures in the bottom or
middle of the tube in the band at the 30 and 42% interface. Resuspend collected
bands in 20 mL of SPGN and pellet at 20,200 ×
g
for 30 min. Resuspend pellet
in SPGN buffer to desired concentration.
3.3. Determining Bacterial Infectious Inoculum
Because of their small size and intracellular location, routine methods
of bacterial enumeration (i.e., Petroff-Hauser chambers) are not possible or
accurate. Two acceptable methods of approximating multiplicity of infection
are as follows:
1. The most common method is to enumerate the number of infected HL60 cells
used to obtain the infectious inoculum and state it as a ratio of number of infected
HL60 cells: number of neutrophils (e.g., to inoculate
A. phagocytophilum
into
neutrophils for a study, state “bacteria liberated from 1 × 10
6
infected HL60 cells
were incubated with 2.5 × 10
5
target cells
(5)
.” Immunofluorescence has been
used to confirm that a ratio of 1 infected HL60 cell : 2 neutrophils is
∼
5-20
A.
phagocytophilum
per neutrophil
(13)
.
2. Alternatively, a formula to estimate multiplicity of infection has been developed:
Estimated number of
A. phagocytophilum
= total infected cell number × average
number of morula in an infected cell (typically 5) × average number of
A.
phagocytophilum
in a morula (typically 19) × percentage of
A. phagocytophilum
recovered as host-cell free [typically 50% as determined by using metabolically
[
35
S] methionine-labeled
A. phagocytophilum
(14)
].
3.4. Bacteria Labeling and Modifications
3.4.1. Labeling with Monoclonal Antibody to Anaplasma
phagocytophilum Conjugated to a Fluorochrome
1. Make a direct or cytofuge smear of the infected cells of interest (e.g., infected
neutrophils or HL60 cells). Air dry.
2. Permeabilize cells with 2 drops of 100% methanol. Air dry.
3. Place several drops of protein block onto slide preparation. Let sit for 10 min.
Remove (dab) excess solution.
4. To conjugate monoclonal antibody: Take 25 μL of R5E4 antibody and add 5 μL of
fluorochrome (reagent A from the Zenon conjugation system). Incubate at room
temperature in the dark for 5 min. Add 5 μL of the blocking antibody (reagent
B from the Zenon conjugation system). Incubate for 5 min. Dilute conjugated
antibody 1:200 in 5% BSA in PBS.
5. Add several drops of conjugated antibody to slide. Incubate the slide for1hin
the dark.
6. Wash with PBS three times for 5 min each time.
7. Fix with 4% PFA in PBS for 20 min.
8. Wash with PBS three times for 5 min each time.
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