Cohort
1:
Lung
single-cell
suspensions
prepared
and
analyzed
Flow
cytometer
Dendritic
cell
counts,
white
blood
cell
counts(subsets)
2
Cohort
2:
Bronchoalveolar
lavage
preformed
and
cells
analyzed
Flow
cytometer
Total
leukocytes,
percent
macrophages
Equipment
and
supplies
Syringes
and
needles
Dissection
kit
20
G
1
1/2
canules
(0.9
x
40
mm)
(BD
GERMANY)
Cell
strainer
70
µM
(BD
GERMANY)
FACS
Canto
II
flow
cytometer
(Becton
Dickinson,
San
Jose
CA)
Reagents
and
solutions
HBSS
(PAA
GERMANY)
Type
A
collagenase
(Roche
GERMANY)
DNase
(Roche
GERMANY)
IMDM
(PAA
GERMANY)
Fetal
calf
serum
(FCS)
(PAA
GERMANY)
Ammonium
chloride
PBS,
2%
BSA,
2
mM
EDTA
(PAA
GERMANY)
PBS,
5%
FBS
(PAA
GERMANY)
PBS,
5
mM
EDTA
(pH
7.2)
Permeabilization
buffer
(ebioscience
GERMANY)
Fluorochrome-labeled
monoclonal
antibodies
(for
full
description
see
Table
1,
Hackstein
et
al.,
2012)
Procedure:
Lung
preparation
Mice
are
euthanized,
and
lungs
are
perfused
via
the
right
ventricle
with
HBSS
to
remove
intravascular
cells.
Lung
tissue
is
minced
and
digested
in
0.09
U/mL
type
A
collagenase
and
9.09
U/mL
DNase
in
IMDM
with
10%
FCS
at
37°C
for
1h.
Single-cell
suspensions
are
prepared
by
tissue
resupension
with
canules
and
by
mashing
through
a
70
µM
cell
strainer.
Red
blood
cells
are
lysed
by
ammonium
chloride.
Cells
are
washed
with
HBSS
for
flow
cytometry
(see
below).
Procedure:
Bronchoalveolar
lavage
(BAL)
Mice
are
euthanized
and
the
trachea
exposed.
A
small
incision
is
made
in
the
trachea
to
insert
a
shortened
21-gauge
cannula
that
is
firmly
fixed
and
connected
to
a
1-mL
syringe
filled
with
300
µL
of
PBS,
5
mM
EDTA
(pH
7.2).
BAL
is
performed
with
300
µL
aliquots
until
a
BAL
volume
of
1.5
mL
is
recovered.
Cells
are
separated
by
centrifugation
and
are
resuspended
for
flow
cytometry
(see
below).
Procedure:
Flow
cytometry
Fluorochrome-labed
monoclonal
antibodies
conjugated
to
FITC,
PE,
PeCy7,
PerCPCy5.5,
APC,
APC-Cy7,
Pacific
Blue,
and
appropriate
isotype
controls
are
used
for
surface
staining
according
to
manufacturer's
instructions
(full
descriptions
of
monoclonal
antibodies,
fluorochrome,
and
source
are
given
in
Table
1,
Hackstein
et
al.,
2012).
Cells
are
stained
for
30
min
on
ice.
Cells
are
washed
with
staining
buffer
(PBS,
5%
FBS)
at
room
temperature.
Cellular
phenotyping
is
performed
on
a
FACS
Canto
II
flow
cytometer
(gating
strategies
are
well
documented
in
Hackstein
et
al.,
2012)