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JPET142950 Supplemental Information
Table 1: Q-PCR TaqMan primers and probe sequences. * house keeping genes and † predetermined target genes.
Gene name
Forward primer
Reverse primer
TaqMan probe
GAPDH*
CAAGGTCATCCATGACAACTTTG
GGGCCATCCACAGTCTTCTG
ACCACAGTCCATGCCATCACTGCCA
B-actin*
GAGCTACGAGCTGCCTGACG
GTAGTTTCGTGGATGCCACAGGACT
CATCACCATTGGCAATGAGCGGTTCC
Cyclophilin* CATCTGCACTGCCAAGACTGA
CCACAATATTCATGCCTTCTTTCA
CCAAACACCACATGCTTGCCATCCA
GMCSF†
AGCCTCACCAAGCTCAAGGG
GGGTTGGAGGGCAGTGCT
CCCTTGACCATGATGGCCAGCC
IL-1β†
AAGCAGAAAACATGCCCGTCT
AATTGCATGGTGAAGTCAGTTATATCCT
CCGCCTTTGGTCCCTCCCAGG
IL-6†
TGACCCAACCACAAATGCCA
CATGTCCTGCAGCCACTGG
CTGTGCCTGCAGCTTCGTCAGCA
IL-8†
TCCTTGTTCCACTGTGCCTTG
TGCTTCCACATGTCCTCACAA
TTAGCCACCATCTTACCTCACAGTGAT
TNF-α†
GGTGCTTGTTCCTCAGCCTC
CAGGCAGAAGAGCGTGGTG
CTCCTTCCTGATCGTGGCAGGCG
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Immunohistochemistry Identification of AM
Alveolar macrophages where plated at 1x105 cells per chamber within cell culture
chamber slides (VWR, Leicestershire, UK). After 24hrs non adherent cells were
discarded and excess media removed with PBS. Cells were fixed in ice-cold methanol
for 10 minutes at -20°C for 10mins. Following 3x5mins washes in PBS, cells were
blocked in 2.5% normal horse serum (NHS) for 30mins at room temperature. Cells
were incubated in a mouse anti-human CD68 antibody (Clone 514H12, Novocastra,
Newcastle, UK) diluted in 2.5% NHS overnight at 4°C. Endogenous peroxidase was
quenched using 3% H2O2 in methanol for 30mins prior to incubation in a peroxidase
conjugated anti-mouse IgG ImmPress kit (Vector Laboratories, Peterborough, UK).
CD68 was visualised using 3,3’-diaminobenzidine tetrahydrochloride (DAB)
chromogen substrate (Vector Laboratories). Cell nuclei were counter stained using
haematoxylin (Sigma, Poole, UK).
Luminex Analysis
Fluorescent microspheres (Luminex Corporation, Austin, Texas, via Applied
Cytometry Systems, Sheffield, UK, Cat No. PN-105-104-01, PN-105-106-01, PN105-107-01, PN-105-108-01) were labelled with primary antibodies to either IL-1β,
IL-6, IL-8 or TNFα. Samples and standards were added to the microspheres in a filter
plate and agitated for 2hrs. Plates were washed with PBS+0.05% Tween 20 (Sigma)
using low vacuum filtration. Biotinylated secondary antibodies were diluted to
0.5ug/ml, mixed with the micropheres and agitated for 1hr. After washing,
streptavidin-PE (PJ31S, Prozyme, San Leandro, CA, USA) was added at 50ug/ml and
the plates agitated for 30mins. Plates were washed, then the micropheres resuspended
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in Luminex sheath fluid (Luminex Corporation via Applied Cytometry Systems, Cat
No. PN-100-006) and the plates read on the Luminex 100 Analyser (Luminex
Corporation).
Antibodies were from Endogen (Endogen, Pierce Biotechnology/Thermo Fisher
Scientific, Rochford, IL, USA, Cat No. M-42OB-B, M620-E, M-621-B, M-801-E and
M-802-B) except for IL-1b and TNFa primaries and the TNFa secondary which were
from R&D Systems (Cat. No. MAB201 (Clone 8516.311), MAB610 and BAF210
respectively). All standards were from R&D Systems. All reagents were diluted in
PBS with 1%BSA (Sigma).
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