Biology Reference
In-Depth Information
1. Grow 5-mL overnight cultures of the wild-type and mutant GAS strains statically
at 37 °C with 5% CO
2
in THY broth.
2. Into four 50-mL tubes, place 40 mL of THY broth and prewarm to 37 °C.
3. For each of the two overnight GAS cultures, perform 1/100 dilutions into two
of the prewarmed 40-mL THY broth aliquots (400 μL of culture into 40 mL
of THY).
4. Incubate cultures statically at 37 °C with 5% CO
2
to the mid-exponential phase
of growth as previously determined by optical density at 600 nm (OD
600
)
measurement.
5. Pellet the bacterial cells by centrifugation (3000 ×
g
for 10 min), wash each
of the pellets twice with 40 mL of PBS, and resuspend each bacterial pellet in
8 mL of PBS.
6. Pool the two cultures corresponding to the same strain, giving 16 mL of washed
wild-type and 16 mL of washed mutant GAS cells.
7. Add 4 mL of 80% glycerol to each 16 mL of GAS solution, mix by inversion,
and aliquot in 2 mL volumes into 15-mL tubes. GAS aliquots are snap frozen in
liquid nitrogen and stored at -80 °C. Approximately 10 aliquots of each strain
should be generated by this protocol.
8. The following day, thaw four of the 10 aliquots of each strain on ice for
30 min.
9. Once thawed, perform serial dilutions of each aliquot in PBS, plate on THY
agar plates, and incubate overnight at 37 °C with 5% CO
2
.
10. The following morning, count colonies and determine the mean (±standard
deviation) colony-forming unit (CFU) titers for the two GAS strains. Typical
titers are
∼
∼
2×10
8
CFUs per mL.
3.2. Cynomolgus Macaque Screens for Group Selection
We commonly use group sizes of four or six cynomolgus macaques per GAS
strain although experiments have been conducted with up to 20 animals
(22)
.
To divide macaques into experimental groups, we use information regarding
animal age, sex, weight, and concentration of serum anti-SLO antibodies. SLO
is a secreted toxin of GAS such that infected individuals seroconvert to SLO
following GAS infection
(24-26)
. Thus, measuring the concentration of anti-
SLO antibodies provides an indicator of previous potential GAS infection. Note
that all animals are tattooed to enable individual identification.
3.2.1. Animal Age, Sex, and Weight
To reduce animal-specific differences in the data, all cynomolgus macaques
should ideally be of the same sex and of similar age (
∼
2-4 years old). It should
be noted, however, that mixed sex experiments and animals ranging in age from
2 to 9 years have previously been used successfully
(22)
. Similarly, differences
in animal weights should be minimalized and taken into consideration during
the grouping process.
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