Project
protocol
-
Contents
Workflow
and
sampling
Step |
Procedure |
Data
collected |
1 |
Mice
euthanized
and
tissues
harvested
(peripheral
blood,
spleen,
thymus) |
- |
2 |
Cell
suspensions
processed
and
analyzed |
Immune
cell
populations |
Equipment
and
supplies
- FACSCalibur
flow
cytometer
(Becton
Dickinson,
San
Jose
CA)
- CellQuest
software
Reagents
and
solutions
- alpha-galactosylceramide
analog
PBS57-loaded
CD1d
tetramers
(NIH
tetramer
core
facility)
- anti-CD16/CD32
(clone
2.4G2)
(BD
Bioscience,
San
Diego
CA)
- anti-CD4
(clone
RM4-5)
(BD
Bioscience,
San
Diego
CA)
- anti-TCR
beta
(clone
H57-597)
(BD
Bioscience,
San
Diego
CA)
- propidium
iodide
Procedure:
Flow
cytometry
- Red
blood
cell-depleted
single
cell
suspensions
are
prepared
from
peripheral
blood,
spleen,
and
thymus.
- Cell
suspensions
are
Fc-blocked
at
room
temperature
with
anti-CD16/CD32
for
10
min
and
washed.
- Cell
suspensions
are
incubated
with
an
antibody
cocktail
containing
CD1d/alpha-galactosylceramide
analog-loaded
tetramers,
anti-CD4
and
anti-TCR
beta
for
30
min
at
4°C.
- Stained
cells
are
washed
and
analyzed
by
flow
cytometry
(dead
cells
are
excluded
by
propidium
iodide).
Data
collected
by
investigator
- Percentage
of
TCR
beta
cells
that
are
invariant
NK
T
(iNKT)
cells
for
peripheral
blood,
spleen,
and
thymus
- Percentage
of
iNKT
cells
that
express
CD4
for
peripheral
blood,
spleen,
and
thymus
Ratios
were
computed
by
MPD.