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  • Kainic acid br Neutrophils or macrophage isolation from bone

    2020-08-30


    Neutrophils or macrophage isolation from bone marrow, spleen, peritoneal cavities and tumors
    To isolate neutrophils from peritoneal cavities, we injected i.p. naive LysMCre+Il1rf/f mice and their wild-type Cre negative controls with 1 mL of 3% thioglycollate broth solution (BD Biosciences) and sacrificed mice in 4-5 hours.
    To isolate neutrophils from spleens and bone marrow we used tumor bearing CDX2ERT-Apcf/f mice with bone marrow from LysMCre+Il1rf/f or control mice. Single cell suspensions were prepared from spleens, flushed tibia and femurs. Cells were filtered r> through 70 mm Cell Strainer (BD Biosciences). For neutrophil purification, Easy Mouse Neutrophil Enrichment kit (StemCell Technol-ogies #19762A) was used for enrichment, followed by FACS sorting for cells with phenotype CD11b+Ly6G+CD4-TCRb-F4/80-.
    Bacteria killing assay
    Sorted neutrophil populations from peritoneal cavities, spleen or bone marrow were pretreated with 100ng/mL of recombinant IL-1b for 1h in complete antibiotic free RPMI media at 37C. Meanwhile, bacteria (suspension of E. coli) were grown to the approximately 1.6 3 108 cells/mL (OD 600 nm = 0.2) or stool bacteria were prepared to similar concentration by disrupting feces in ice cold PBS and filtering through 40 mcm strainer. Next, E.coli or ‘‘bulk’’ stool bacteria was incubated for 30 min with 20% of normal mouse serum for opsonization and after that added to neutrophils in the approximate of 50:1 bacteria/neutrophil ratio, incubated for 1 h at 96-well plate, then spin down for 5 min at 1500 rpm and lysed in DI water with 0.1% Triton-100. Lysates were diluted serially (1:10, 1:100, 1:1000) for plating on LB or blood agar. Plates were incubated in Kainic acid (BD BBL GasPak Anaerobic Systems) or regular aerobic conditions overnight at 37C. Colonies were counted manually.
    Phagocytosis assay
    Sorted neutrophil populations from peritoneal cavities or spleens were pretreated with 100ng/mL of recombinant IL-1b for 1 h in com-plete RPMI media at 37C, then suspension of zymosan beads conjugated with Alexa Fluor 594 dye (Invitrogen) or Salmonella-GFP was added and cultured with neutrophils for 2h. Cell suspensions were transferred on Poly-L-lysine coated glass slides, fixed with 4% PFA for 5 min and stained with anti-Ly6G (A18)-FITC or -APC antibodies in 1:75 ratio for 20 min and counterstained with DAPI for confocal imaging.
    Microbiome analyses
    Bacterial DNA was isolated from a fecal content or epithelial ‘‘shakes’’ of various naive or CRC bearing mouse strains. Samples were homogenized with RNase/DNase free 2.8mm Ceramic Beads using Omni Bead Ruptor 24 in ASL buffer (Stool lysis buffer; QIAGEN) followed by DNA isolation using QIAamp DNA stool Mini Kit (QIAGEN) according to manufacturer’s protocol. Library preparation, sequencing and data analysis were carried out essentialy as previously described (Fatkhullina et al., 2018). Briefly, QPCR was carried out using universal or bacterial strain-specific primers for 16S rRNA genes. The V4 region of the 16S rDNA gene using two round amplification PCR using Phusion High-Fidelity DNA-polymerase (NEB) with 100ng of input DNA. First round PCR (15 cycles): 515F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG
    806R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC Second round PCR (10 cycles) with i5 and i7 Illumina barcode sequences: AATGATACGGCGACCACCGAGATCTACAC-i5-TCGTCGGCAGCGTC CAAGCAGAAGACGGCATACGAGAT-i7-GTCTCGTGGGCTCGG
    Amplicones were purified between and after the PCR reactions with Agencourt AMPure XP (Beckman Coulter, Inc.) and libraries were quantified using KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems) and size was estimated using Agilent Tapestation 4200 (Agilent). Libraries were pooled at equimolar concentration using Epmotion 5075 tmx automatic liquid handling machine (Eppendorf) and sequenced with 5% PhiX library added to the pool on Illumina MiSeq machine according to manufacture instructions using v3 kit (600 cycles). Fastq files were generated on Basespace (Illumina). On average 100,000 reads were obtained per sample, and after quality control filtering; on average 90,000 reads per sample were processed further. Chimeric sequences were filtered out of the FASTQ files containing the 16S rRNA gene sequences using the USEARCH (version 8.1) utility’s UCHIME implementation and the ‘gold’ database (version microbiomeutil-r20110519). The reads, thus filtered, were then binned into operational taxonomic units (OTUs) at 97% similarity using USEARCH’s cluster_otus command. QIIME (1.9.1) scripts were used to classify and align the obtained OTUs. The assign_taxonomy.py script was used to assign taxonomy using the default RDP method and GreenGenes database.