CHAPTER 2: MATERIALS AND METHODS
2.9 Animal studies
2.9.1 Mice housing
DUSP6-null mice (B6;129X1-Dusp6tm1Jmol/J,57 stock number 009069, backcrosses
number=1) and their appropriate control mice (B6129SF2/J, stock number 101045, in
19
recommended by the manufacture http://jaxmice.jax.org/strain/009069.html) were
purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were bred and
maintained in a specific pathogen-free (SPF) animal facility in a room subjected to a
12-hours light/dark cycle and maintained at constant temperature (22ɗ) and humidity
(55%). Mice received normal rodent chow and water ad libitum. All experimental
procedures were performed in accordance with the guidelines of the Institutional
Animal Care and Utilization Committee (IACUC) of Academia Sinica.
2.9.2 Genotyping
The genomic DNA was extracted from the tail tissue of mouse by the KAPA Express
Extract kit (KAPK Biosystem) according to the manufacturer’s instructions. A common
forward primer A (5’-CCT TCT CCT GCA GCT CGA C-3’, #12227), the wild type
mouse reverse primer B (5’-ATG GCA GAT TCG ATG TGT GA-3’, #12226) and
Dusp6-/- mouse reverse primer C (5’-CCG CTT CAG TGA CAA CGT C-3’, #12228,
catalog numbers provided by The Jackson Laboratory) were used for standard PCR in a
mixture of the KAPA2G Robust HotStart reagent (KAPK Biosystem) according to
manufacturer’s instruction.
0 0 0 0 0 0 0 0 0 0 0 0 0
09090909090909090909090909069000000006969696969696969696696966666969.h99.h.h.h.htmtmtmtmtmlll) ) ) wewewewewereeeeeererererrererrererrrrrrrreeee
ceeeeeeeeeeeeeeeee wwwwwwwwwwwwwwwwwererererereeeeee e e e eee brbrbrbrbrbrededededed aaaaandndndndndndndndndndndndndndndnnndddd
om subjbjbjbjbjbjbjbbjbjjeececececeeecececcteteteteeeeeeeddddddddddddddd to a
20
2.9.3 Tail vein injection with TNF-DD
In order to induce endothelium inflammatory response, male mice (10-12 weeks old) were injected with 5 Pg/kg of TNF-D diluted in PBS (Sigma) to a total volume of 100
Pl) into the lateral tail vein. Control mice were injected with an equal volume of PBS.
After 16 hours, mice were sacrificed. Vessels (containing aorta and inferior vena cava
(IVC)) were removed and processed for immunohistochemical staining.
2.9.4 Immunohistochemstry staining and image quantification
Organs from TNF-D-, LPS- or PBS-treated mice were harvested, rinsed in ice-cold
PBS, fixed in 4% paraformaldehyde and then embedded in paraffin. For
immnunohistochemistry staining, tissue sections were blocked with 10% goat serum
(005-000-001, Jackson Immunoresearch) for 2 hours and then incubated for overnight
with anti-mouse ICAM-1 antibodies (14-0542) or isotype control (14-4321, both from
eBioscience) at a dilution of 1:50. After three washes in PBS, the samples were treated
with goat anti-rat IgG secondary antibody (A9037, Sigma) at a dilution of 1:200 for 1.5
hours at room temperature. Bound antibody was detected using a DAB kit (Vector
Laboratories). Sections were counterstained with hematoxylin and eosin (H & E, both
from Sigma-Aldrich), dehydrated, treated with xylene substitute (Fluka) and
subsequently mounted with entellan (Merck). Images of the whole aorta and IVC were (1
21
captured using a microscope (BX50, Olympus) with 60x magnification, and images of
lung were captured with 40x magnification. For quantification, images were processed
and analyzed using software Image-Pro Plus 6.0 (Media Cybernetics).
2.9.5 LPS-induced experimental sepsis and neutrophil adoptive transfer
For induction of the experimental sepsis, male mice (5-8 weeks old) were injected intraperitoneally with 0.1 mg/kg of TNF-D or 10 mg/kg of LPS in a total volume of 200
Pl. After 24 hours, mice were sacrificed. Lung were isolated and processed for
myeloperoxidase (MPO) determination by MPO-specific enzyme-linked
immunosorbent assay (ELISA; HyCult Biotechnology) according to manufacturer’s
instruction. For neutrophil adoptive transfer, male mice (10-12 weeks old) were utilized.
The endogenous polymorphonuclear leukocytes (PMNs, mainly neutrophils) of
recipient mice were removed by irradiation (9 Gy) exposure. After recovery for 24
hours, 1x107 purified neutrophils in PBS (total volume 200 Pl) were adoptively
transferred to recipient mice by intravenous injection, followed by intraperitoneal injection with 10 mg/kg of LPS in a total volume of 200 Pl. After 4 hours, mice were
sacrificed. Lung were isolated and processed for H&E staining, immunohistochemistry
staining with anti-ICAM-1 antibody and MPO assay.
ioooooooooooooooon,n,n,nn,n,n,n,n,n,n,n,n,n,n,n,n,nn,n, aaaaaaaaaaaaaaaaandndndndnd iiiiimamamamamagegegegegess sss ofofofofofofoofofoofoooooooofff
geeeeeeeeeeeeeeeeessssssssssssss wewewewewewewewwewwwwwewwewewewwww rerereree prprprprrrocooooocesesesesese seseseseseeeeeeeeeeeeeedddd d dd d d dd dddd dddd
22
2.9.6 Neutrophil isolation from mouse blood
The procedure of neutrophil isolation was performed according to the protocol
described previously.58 In brief, whole blood from adult donor mouse was collected in
tubes containing EDTA and then mixed with an equal volume of PBS. The cells were
separated onto a three-layer Percoll gradient of 78, 69, and 52% Percoll diluted in PBS
through centrifugation at 1500x g for 35 min at room temperature. The fraction of
neutrophils at the 69/78% interface were harvested and washed with PBS containing 1%
BSA once. The residual red blood cells were then eliminated by RBC Lysis Buffer
(Becton Dickinson) at 37ɗ for 3 min. After two times of wash with PBS containing
1% BSA, the purified neutrophils were suspended in PBS and used immediately. The
purity and viability of purified neutrophils was confirmed by Ly6G/CD11b double
staining and trypan blue (Sigma) exclusion, respectively.
2.9.7 Flow cytometry analysis
Cells were incubated with Ly6G-FITC (11-5931), CD11b-PerCP-Cyanine5.5
(45-0112) or isotype control antibodies (11-4031 and 45-4031, all from eBioscience)
against cell surface antigens in the dark for one hour on ice. Cytofluorimetry was
performed with a BD Calibur cytometer (Becton Dickinson) equipped with FL1
(533/30), FL3 (650LP) filters. Neutrophils were identified by characteristic forward/side g
23
scatter and Ly6G/CD11b positivity. Data were analyzed and presented using the BD
CellQuest Pro software (Becton Dickinson).