FL cell isolation and sorting

Transcription

1 FL cell isolation and sorting Mononuclear cells obtained from FL were blocked with an anti-cd16/32 (93) antibody (BioLegend), followed by predepletion of differentiated hematopoietic cells with automacs TM (Miltenyi Biotec) using the peridinin-chlorophyll-protein complexcyanine 5.5 (PerCP-Cy5.5)-conjugated lineage markers rat anti-cd4 (RM4-5), anti- CD8 (53-6.7), anti-b220 (RA3-6B2), anti-gr-1 (RB6-8C5), and anti-ter119 (Ter-119) antibodies (BD Biosciences), as well as goat anti-rat IgG microbeads (Miltenyi Biotec). Lineage cells were further stained with biotinylated anti-epcr (RMEPCR1560) (Stemcell Technologies), fluorescein isothiocyanate (FITC)-conjugated anti-c-kit (2B8), and phycoerythrin (PE)-conjugated anti-sca-1 (D7) (BD Biosciences) antibodies, followed by incubation with APC-conjugated streptavidin (BD Biosciences). Finally, propidium iodide (Invitrogen) was added to discriminate dead cells. A BD FACSVantage TM system and the FACSDiva TM software (BD Biosciences) were used to perform flow cytometry analysis and cell sorting. Immunohistochemistry Embryos were fixed in 2% paraformaldehyde in phosphate buffer solution (PBS) for 5 h at 4ºC and washed in PBS overnight. After 30% sucrose infusion, embryos were embedded in OCT compound (Sakura Finetek Japan) and frozen. Frozen embryos were sectioned at a thickness of 12 µm and transferred onto a silanized glass slide (Dako). Sections were then washed in PBS and endogenous peroxidase was quenched with 0.3% H 2 O 2 in PBS for 30 min, followed by blocking with 1% bovine serum albumin in PBS. Endogenous biotin was blocked using the Avidin/Biotin Blocking Kit (Invitrogen). Sections were incubated overnight at 4ºC in PBS with selected primary antibodies: biotinylated rat anti-epcr (Stemcell Technologies), rabbit anti-mouse lymphatic vessel endothelial receptor 1 (LYVE-1) (103-PA50AG, ReliaTech), goat anti-mouse LYVE-1 (AF2125, R&D Systems), goat anti-mouse c-kit (AF1356, R&D Systems), rabbit antimouse alpha laminin (L9393, Sigma Aldrich), rabbit anti-human fibronectin (A0245, Dako), rabbit anti-mouse matrix metalloproteinase 9 (MMP-9) (AB19047, Millipore), goat anti-mouse TM (AF3894, R&D Systems), rat anti-human APC (HyCult Biotech), and/or hamster anti-mouse delta-like 1 homolog (DLK-1) (kindly provided by Kyowa Hakko Kirin). After washing, biotinylated rat anti-epcr staining was amplified using the TSA Biotin System (PerkinElmer Life And Analytical Sciences). Sections were then stained with secondary antibodies: Alexa Fluor 488-conjugated streptavidin, goat anti-rabbit IgG, or donkey anti-rabbit IgG (Invitrogen); Cy3-conjugated goat anti- Armenian hamster IgG, donkey anti-rabbit IgG, or donkey anti-goat IgG (Jackson ImmunoResearch Laboratories); Alexa Fluor 633-conjugated streptavidin (Invitrogen); or Cy5-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Nuclei were stained with TOTO-3 or 4',6-diamidino-2- phenylindole (DAPI) (Invitrogen). For costaining with lineage markers, sections were stained overnight first with anti-epcr and goat anti-mouse Sca-1 (AF1226, R&D Systems) antibodies, followed by first secondary antibody staining with Cy3-conjugated donkey anti-rat IgG (Jackson ImmunoResearch Laboratories) and Cy5-conjugated donkey anti-goat IgG. Subsequently, they were stained with second primary antibodies hamster anti-mouse DLK-1 and rabbit anti-mouse Lyve-1. Finally, second secondary antibodies FITC-conjugated goat anti-armenian hamster IgG (Jackson

2 ImmunoResearch Laboratories) and DyLight 405-conjugated goat anti-rabbit IgG (Thermo Scientific) were used for labeling along with lineage staining using FITCconjugated rat anti-cd4, anti-cd8, anti-b220, anti-gr-1, anti-ter-119, anti-cd41 (MWReg30, BD), and Armenian hamster anti-cd48 (HM48-1, BioLegend) antibodies. Cell cycle analysis For cell cycle analysis using flow cytometry, pregnant mice were injected intraperitoneally with 5-Bromo-2-deoxyuridine (BrdU) (5 mg each, Sigma Aldrich) 2 h prior to being sacrificed. FLs were dissected and differentiated hematopoietic cells were depleted as described in Methods. After lineage cell depletion and surface-marker staining, cells were fixed and permeabilized using the BrdU Flow Kit (BD Bioscience). DNA was cleaved with a 1 h treatment of 0.3 mg/ml DNase (Sigma Aldrich) at 37ºC and incorporated BrdU was stained subsequently with an anti-brdu antibody. Nucleic acids were stained with Hoechst trihydrochloride trihydrate (Invitrogen). Additionally, Ki67 analysis was performed to assess G0 phase fraction. After lineage cell depletion and surface marker staining, cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Bioscience), followed by staining with an Alexa Fluor 488-conjugated anti-ki67 antibody (BD Bioscience). RNA was digested with 50 µg/ml RNase A for 20 min in a water bath at 37ºC.

3 Table S1. List of mrnas investigated by high-throughput qrt PCR analysis Gene ID Assay ID Gene ID Assay ID Gene ID Assay ID Gene ID Assay ID Alcam Mm _m1 cdh4 Mm _m1 Flt1 Mm _m1 Notch2 Mm _m1 Angpt1 Mm _m1 cdh5 Mm _m1 Flt3 Mm _m1 Pbx1 Mm _m1 Aurka Mm _m1 cdh6 Mm _m1 Flt4 Mm _m1 Pcdh7 Mm _m1 Bax Mm _m1 cdh7 Mm _m1 Gata1 Mm _m1 Pdgfa Mm _m1 Bbc3 Mm _m1 cdk2 Mm _m1 Gata2 Mm _m1 Ppp1r13b Mm _m1 Bcl2 Mm _m1 cdk4 Mm _s1 Gata6 Mm _m1 Procr Mm _m1 Bmi1 Mm _gH cdk6 Mm _m1 Gfi1 Mm _m1 Psg1-1 Mm _m1 Bmp2 Mm _m1 Cdkn1a Mm _m1 Hoxb4 Mm _m1 Ptgs2 Mm _m1 Bmp4 Mm _m1 Cdkn1b Mm _g1 Icam1 Mm _m1 Runx1 Mm _m1 Bmp6 Mm _m1 Cdkn1c Mm _g1 Itga4 Mm _m1 Sall4 Mm _m1 Ccnd1 Mm _s1 Cdkn2a Mm _m1 Itga9 Mm _m1 Slamf1 Mm _m1 Ccnd2 Mm _m1 Cdkn2b Mm _m1 Itgal Mm _m1 Sox17 Mm _m1 Ccnd3 Mm _m1 Cdkn2c Mm _m1 Itgb1 Mm _m1 Sox2 Mm _s1 Ccne1 Mm _m1 cdx2 Mm _m1 Itgb2 Mm _m1 Tal1 Mm _m1 Ccne2 Mm _m1 Csf3r Mm _m1 Kdr Mm _m1 Tcf3 Mm _m1 Ccng2 Mm _m1 ctnna1 Mm _m1 Kit Mm _m1 Tek Mm _m1 Cd44 Mm _m1 ctnnb1 Mm _m1 Mapk1 Mm _m1 Tert Mm _m1 Cdh1 Mm _g1 Ctnnd1 Mm _m1 Mapk3 Mm _g1 Trp53 Mm _g1 Cdh11 Mm _m1 Cxcr4 Mm _m1 Mcam Mm _m1 Vcam1 Mm _m1 Cdh12 Mm _m1 Ecat1 Mm _g1 Mpl Mm _m1 Vegfa Mm _m1 cdh15 Mm _m1 Eng Mm _m1 Myc Mm _m1 Wnt5a Mm _m1 cdh2 Mm _m1 Etv6 Mm _m1 NfkB Mm _m1 Wnt5b Mm _m1 cdh3 Mm _m1 Evi1 Mm _m1 Notch1 Mm _m1

4 Figure S1 (A D) Tcf3, Itga4, Itga9, and Gfi1 were screened based on their significantly different expression levels between LSK/EPCR + and EPCR cells or on the significant changes observed for these genes in EPCR + cells during 2-day short culture. Dark gray bars, LSK/EPCR + cells. Light gray bars, LSK/EPCR cells. Error bars represent means ± SD (n = 2). P < 0.1, *P < 0.05.

5 A Tcf3 B Itga4 C D Itga9 * Gfi1