2.6.1 Lymph nodes cell preparation and stimulation.
Skin draining lymph nodes were collected from the axillas of each mouse
and put into the precooled wash buffer (2 % FBS and 0.2 % 0.5 M EDTA
in 1*PBS). All lymph nodes were ground with the frosted surface of glass
slides and passed through 70 μ m strainers to generate the
single-cell suspension.
2.6.2
Epidermics cell preparation and stimulation.
The skin on the back of mice (2 cm*2 cm) was cut and placed in a 60-mm
petri dish with PBS buffer and placed on ice. The excess subcutaneous
tissue under the skin was gently scraped off with a scalpel blade,
washed once with 75% ethanol quickly, and washed twice with PBS. The
epidermis was placed in a 60mm petri dish with 0.25% dispase (0.025g
dispase+10 mL 1*PBS), incubated at 37℃ for 1h, and then the dermal part
of the skin was gently scraped off. After the epidermis was cut into
pieces, 700 μ L DNase (1 mg/mL) and 6.3 mL 1 *PBS were added and
incubated at 37℃ for 50-60min. Digestion was terminated with a small
amount of staining buffer(2 % FBS in 1*PBS), and epidermal cells were
collected and counted after filtration with 100 μ m and 40μ m filters.
2.6.3 Flow cytometric detection of DC and LC in lymph
nodes and epidermis. One million cells per
staining were incubated with BD Pharmingen™ Purified Rat Anti-Mouse
CD16/CD32 (Mouse BD Fc Block™) (clone: 2.4 G2, BD) for five minutes to
block non-antigen-specific binding of immunoglobulins. Then cells were
stained for cell surface and intracellular markers with the following
conjugated monoclonal antibodies:
MHC
Class II (I-A/I-E) Monoclonal Antibody, eFluor™ 450 (clone:
M5/114.15.2,
eBioscience), CD207 (Langerin) Monoclonal Antibody, PE (clone: eBioL31,
eBioscience), Alexa Fluor® 647 anti-SPI1 (PU.1) Antibody (clone: 7C2C34,
Biolegend),
BD
Pharmingen™ FITC Rat Anti-Mouse CD103 (clone: M290, BD Pharmingen),
BD
Pharmingen™ Alexa Fluor® 647 Rat Anti-Mouse CD45R (clone: RA3-6B2, BD
Pharmingen), BD Pharmingen™ PerCP-Cy™5.5 Rat Anti-CD11b (clone: M1/70).
Cells were analyzed with FACS Aria Ш (BD Biosciences, San Jose, CA, USA)
and FlowJo software version 10.0 for Microsoft (TreeStar, Sam Carlos,
CA).