Supporting Information
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Supporting Information Iftakhar-E-Khuda et al. 10.1073/pnas.1602357113 SI Methods P (Roche) and 0.8 mg/mL neutral protease (Worthington Mice. C57BL/6J (stock 000664) WT, and MSR KO (B6.Cg- Biochemical) for 45 min at 37 °C. The isolated cells were cul- tm1Csk tured in gelatin-coated flasks in complete mouse endothelial cell Msr1 /J (stock 006096) mice were purchased from The − − medium/w kit (Cell Biologics) for 7 d. Thereafter, CD45 /CD11b / Jackson Laboratory. All animal experiments were approved by + + + the Ethical Committee for Animal Experimentation in Finland. podoplanin /CD31 /Lyve-1 cells were low-pressure sorted using FACSAria IIu fitted with a 100-μm tip. Sorted LECs were seeded Antibodies. Detailed information of the antibodies used is given in at a concentration of 5 × 105 cells/cm2 andculturedfor7d. Table S3. Microarray Analysis. RNA amplification, labeling, and cDNA LCMP. Inguinal, axillary, brachial lymph nodes (LNs) from C57BL/ microarray hybridization and gene expression analyses were 6J mice aged 8 wk were used for the isolation of LECs from the SS performed at the Miltenyi Biotec Genomic Services (Miltenyi and LS. LNs were collected in Rnase-free phosphate buffer saline Biotec). The sorted cells in SuperAmp Lysis Buffer were sub- containing 1 unit/μL SUPERase-In (Applied Biosystem) and jected to SuperAmp RNA amplification based on a global PCR thereafter, placed onto a layer of OCT containing 20 μg/mL protocol using mRNA-derived cDNA according to Miltenyi vanadyl ribonucleoside complex (Sigma-Aldrich) solution. More Biotec’s undisclosed procedure. The integrity of the cDNA was solution was added over the LNs until they were completely checked with the Agilent 2100 Bioanalyzer platform (Agilent covered. The filled cryomolds were immediately frozen in dry-ice Technologies). The average length of the highly amplified cDNA chilled isopentane and stored in a −70 °C freezer until use. products ranged between 200 and 1,000 bp. The cDNAs (250 ng) Alexa Fluor 488 anti-mouse CD31 (Biolegend) and PE-Texas were used as templates for Cy3 labeling using Miltenyi Biotec’s red anti-mouse F4/80 (Invitrogen) antibodies were applied onto undisclosed protocol. The Cy3-labeled cDNAs were hybridized the frozen sections of the LNs for 3 min at 4 °C. After washing, overnight (17 h, 65 °C) to Agilent Whole Mouse Genome Oligo the sections were dehydrated through ascending concentrations Microarrays 8× 60 K using Agilent’s recommended hybridization of ice cold ethanol [70%, 95%, and 100% (vol/vol)] for 10 s each chamber and oven. Fluorescence signals of the hybridized Agi- and finally in xylene for 10 s. lent Microarrays were detected using Agilent’s Microarray Scanner Microscopic observations of the cells were performed using a System (Agilent Technologies). The Agilent Feature Extraction Zeiss microscope with filter set 25 (Zeiss) and LCMP was con- Software was used to read out and process the microarray image ducted on a PALM microbeam instrument including Palm-Robo files. The software determines feature intensities (including back- version 3 software. To maximize the collection of the right cells, in ground subtraction), rejects outliers, and calculates statistical + the SS only discrete CD31 cells were collected. In the LS area, confidences. The microarray data produced in this study has been the blood vessels stain much brighter for CD31 than do LECs. deposited to GEO archive (GSE 68371). Moreover, LS lymphatics can be morphologically discriminated In bioinformatics analyses, the intensity data were background from the blood vessels. SS and LS endothelial cells were col- corrected and quantile normalization was conducted between the lected based on these characteristics using a 40× objective and arrays. The normalized intensities were log2 transformed and the fluorescence filter. LCMP of the labeled cells was performed used as a basis for further analysis. Interexperiment correlation under bright-field optics. The following settings were used for analysis of normalized log2 intensities was performed using Eu- the microdissection procedure: dissection method, Robo LPC; clidean distance metric. The differential gene expression be- UV energy, 50–65; UV focus, 45–50; and cutting speed, 20. tween the SS and the LS of the LCM samples was tested with a Microdissected cells were collected to adhesive caps (Zeiss); one-way ANOVA and an adjusted P value was calculated by the before their use, the caps were irradiated with UV (254 nm) for method of Benjamini and Hochberg (24). All samples of the SS 30 min. Five cells were isolated per slide to minimize the time and LS populations regardless of the isolation method were (15–20 min) and maximize the quality of RNA. Altogether 50 further used as a test and reference group, respectively, by per- cells were collected per experiment. The cells were collected forming Student’s t test (two tailed). The differentially expressed into the superamp lysis buffer (Miltenyi Biotec), incubated at 45 °C (DE) genes were selected requiring an absolute fold-change >2 for 10 min, and thereafter stored at −40 °C. and a P < 0.05. For stringent selection, the magnitude of the expression difference was at least eightfold. A detection P value Isolation and Sorting of LECs. Peripheral LNs and MLNs from 12 (Rosetta resolver error model) (25) was also calculated for each mice were pooled and digested in a freshly prepared enzyme mix reporter to indicate whether the signal is reliable or not. This [PBS containing 25 μg/mL liberase TM research grade (Roche) P value was used in further filtering of the data to allow only and 100 μg/mL DNase I (Roche)]. For cell sorting, the following records with the majority of samples fulfilling the detection cri- antibodies were used: APC-conjugated lineage (CD3e, CD11b, teria (detection P < 0.01) in the group with higher expression. CD45R/B220, erythroid cells, Ly-6G, and Ly-6C) mixture, with Functional grouping analyses were performed using the dif- an isotype control (BD Pharmingen), PE anti-CD73 (BD Phar- ferentially expressed genes as input gene populations. The an- mingen), and PE/Cy7 anti-podoplanin (Biolegend). Seven-AAD notations used were derived from Gene Ontology databases (Calbiochem) was used to exclude dead cells. The 106 cells/100 μL and various other pathway resources curated by Miltenyi Biotec. were incubated with 1 μL of each antibody for 15 min at 4 °C The differentially expressed reporters were tested for significant before acquisition on a FACSAria IIu (BD Bioscience). A total of enrichments of annotations using the TreeRanker software 1,200 cells were sorted into FCS-coated FACS tubes con- (Miltenyi Biotec). The frequency of the association of a category taining RPMI-1640 and 20% (vol/vol) FCS; cell purity was with the input reporter set was compared with that of a back- more than 98%. The gating was always done based on the ground set (Agilent 8× 60 K array genes). P values were com- stainings with negative control antibodies. puted by Fisher’s exact test with Benjamini–Hochberg correction For primary cell cultures, mouse peripheral and mesenteric for multiple testing. Values ≤0.05 indicate a significant enrichment LNs were digested with PBS containing 0.2 mg/mL collagenase of the respective category relative to the background (all reporters Iftakhar-E-Khuda et al. www.pnas.org/cgi/content/short/1602357113 1of10 with a gene ID (Entrez) of the Agilent Whole Mouse Genome Flow Cytometric Analyses. Enzyme digestions and antibody staining Oligo Microarray 8× 60 K). The data were further analyzed using of LNs were performed as described in Isolation and Sorting of GENE-E analysis platform (https://www.broadinstitute.org/cancer/ LECs. The skin was digested for 45 min in a freshly prepared software/GENE-E/). enzyme mix (PBS containing 8 mg/mL collagenase IV from Sigma and 100 μg/mL DNase from Roche). The following pri- Immunohistochemical Analyses. Frozen sections were stained with mary, secondary, and isotype-control antibodies were used for primary antibodies: rat anti-mouse MSR1/CD204 (Lifespan stainings: APC-Cy7 rat anti-mouse CD45 (BD Pharmingen), Bioscience), rat anti-mouse Siglec-1/CD169 (AbD Serotec), rat APC-Cy7 rat anti-mouse CD11b (BD Pharmingen), PE/Cy7 Sy- anti-mouse EMCN (EMD Millipore), rabbit anti-mouse Lyve-1 rian hamster anti-mouse podoplanin (Biolegend), APC rat anti- (ReliaTech), Syrian hamster anti-mouse podoplanin/gp36 (Abcam), mouse CD31 (Biolegend), PE-conjugated rat anti-mouse Lyve-1 and rat anti-mouse PLVAP-1 (Meca 32, gift from Eugene Butcher, (R&D Systems), PE rat IgG2A κ-isotype control antibody (BD Stanford University, Stanford, CA). The second stage reagents Pharmingen), rat anti-mouse CD204 (Lifespan Bioscience), rat were: Alexa Fluor 546 goat anti-rat IgG (H+L) (Invitrogen), Alexa anti-mouse CD169 (AbD Serotec), rat anti-mouse EMCN (EMD Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen), Alexa Fluor Millipore), eFluor 660 anti-mouse EMCN (eBioscience), FITC- 546 goat anti-rabbit IgG (H+L) (Invitrogen), and Alexa Fluor 488 conjugated rat IgG1 λ-isotype control (Biolegend), FITC-conjugated goat anti-hamster IgG (H+L) (Invitrogen). The following direct rat IgG2A κ-isotype control (BD Pharmingen), FITC-conjugated rat conjugates were used: Alexa Fluor 488-conjugated rat anti-mouse IgG2B κ-isotype control (BD Pharmingen), and FITC-conjugated CD204 (BIO-RAD) and Alexa Fluor 488-conjugated rat anti- goat anti-rat IgG (whole molecule) (Sigma). Cells were analyzed with mouse Lyve-1 (R&D Systems), Alexa Fluor 647 rat anti-mouse FACSAria IIu and LSR Fortessa (both BD Biosciences) using CD4 (BD), Pacific Blue rat anti-mouse CD45R (BD), and Alexa FlowJo software (Tree Star). Fluor 488 rat anti-mouse CD8 (BD) or Alexa Fluor 488 rat anti- mouse CD31 (Biolegend). Finally, the sections were mounted in Adhesion Assays. Ex vivo. ProlongGold anti-fade reagent (Molecular Probes) and images In principle the adhesion assays were performed as de- were acquired using an Olympus BX60 microscope or an LSM 780 scribed earlier (26).