Cowell1 project protocol
Host response to Staphylococcus aureus infection in females of 13 inbred strains of mice (2012)
Cowell LG, Fowler VG
With: Johnson NV, Ahn SH, Deshmukh H, Levin MK, Nelson CL, Scott WK, Allen A
Cowell1
Protocol
Project
protocol
-
Contents
Workflow
and
sampling
Step |
Procedure |
Data
collected |
1 |
Cohort
1:
Injected
with
S.
aureus
for
survival
study
(max
104
h) |
Survival
time
(
in
hours) |
2 |
Cohort
2:
Injected
with
S.
aureus
for
determining
bacterial
load
(at
24h) |
colony
forming
units
(kidney
and
peritoneal
fluid) |
3 |
Cohort
3:
Injected
with
S.
aureus
for
determining
serum
IL-6
levels
(at
24h;
compared
to
uninjected
controls) |
IL-6
levels |
Equipment
and
supplies
- Syringes
and
needles
- Dissection
kit
- Trypic
Soy
Agar
plates
Reagents
and
solutions
- Phosphate
buffered
saline
(PBS)
- Bicinchoninic
acid
method
kit
(Pierce)
- ELISA
kit
(Due
Kit,
Invitrogen)
Procedure:
Bacterial
preparation
and
injection
- Sanger
476
strain
of
Staphylococcus
aureus
is
used.
- Mice
are
injected
intraperitoneally
with
107
colony
forming
units
per
gram
(CFU/g)
of
S.
aureus.
Procedure:
Survival
study
- S.
aureus
injected
mice
are
monitored
every
8h
for
5
days.
- Mice
are
euthanized
using
CO2
asphyxiation
if
they
appear
moribund.
- Survival
times
are
recorded
in
hours
(max
104
h).
Procedure:
Bacterial
load
- S.
aureus
injected
mice
are
euthanized
24h
post-infection.
- Kidneys
are
collected
and
homogenized
in
PBS;
diluted
serially
with
PBS
10-fold.
- Peritoneal
fluid
is
collected;
diluted
serially
with
PBS
10-fold.
- Serial
dilutions
(50
µL)
are
plated
on
Tryptic
Soy
Agar
plates
and
incubated
overnight
at
37°C.
- Bacterial
colonies
are
counted
(CFU/g
for
kidney;
CFU/mL
for
peritoneal
fluid).
Procedure:
Serum
IL-6
levels
- S.
aureus
injected
mice
are
euthanized
24h
post-infection.
- Blood
is
obtained
by
intracardiac
puncture.
- Serum
is
separated
by
centrifugation.
- Total
protein
is
determined
using
the
bicinchoninic
acid
method.
- Serum
is
diluted
to
ensure
equal
amounts
of
total
protein.
- IL-6
levels
are
estimated
using
an
enzyme-linked
immunosorbent
assay
(ELISA).
- Fold-change
is
determined
compared
to
uninfected
mice
of
the
same
strain.
Data
collected
by
investigator
- Survival
(hours
post-infection;
max=104
h)
- Bacterial
load:
kidneys
- Bacterial
load:
peritoneal
fluid
- Serum
IL-6
levels
(fold-change
compared
to
uninfected
controls)