Isolation and cryopreservation of leukocytes:<\/strong><\/em><\/p>\n Basic Microbiology:<\/em><\/p>\n Streptococcus pneumoniae:<\/em><\/p>\n Puchta A, Verschoor CP, Thurn T, Bowdish DM.\u00a0Characterization of inflammatory responses during intranasal colonization with <\/a>Streptococcus pneumoniae.<\/a>\u00a0<\/em>J Vis Exp. 2014 Jan 17;(83):e50490. doi: 10.3791\/50490.<\/p>\n<\/div>\n<\/div>\n Tissue collection<\/span>:<\/em><\/p>\n![\"\"](\"https:\/\/www.bowdish.ca\/lab\/wp-content\/uploads\/2011\/07\/Macrophage-bullet.gif\" "\\"Macrophage-bullet\\"")
<\/a>\u00a0<\/em>Yearly check-in \u00a0(undergraduate)<\/a><\/p>\n\n
<\/a><\/em>Yearly check-in (graduate)<\/a><\/p>\n\n
\n
<\/a><\/em>Guidelines for Laboratory notebooks and data<\/a><\/p>\n\n
<\/a>\u00a0<\/em>Bowdish lab exit checklist<\/a><\/p>\n\n
General Protocols:<\/em><\/h2>\n
<\/a><\/em>How to Write your first Manuscript<\/a><\/p>\n<\/a><\/em>How to do a journal club<\/a>.<\/p>\n\n
<\/a>Cytokine ELISA\u00a0<\/a><\/p>\n\n
How to titrate antibodies for DIY ELISAs<\/a><\/p>\n\n
Methylene Green SDS Detection Assay<\/a><\/p>\n\n
Protein A Sepharose purification of Antibodies<\/a><\/p>\n\n
<\/a>Isolation of\u00a0peripheral blood mononuclear cells (PBMC) using Leucosep tubes and cryopreservation<\/a>.<\/em><\/p>\n<\/a>Isolation of\u00a0peripheral blood mononuclear cells (PBMC) and granulocytes using a Ficoll gradient and subsequent cryopreservation<\/a>.<\/em><\/p>\n\n
Molecular Biology:<\/em><\/h2>\n
Cloning<\/a>
\n<\/a><\/p>\nThe PCR resource manual.<\/a><\/p>\n\n
<\/a> <\/em>Designing primers for qPCR<\/a><\/p>\nImmunoprecipitation<\/a><\/p>\n\n
Co-immunoprecipitation<\/a><\/p>\n<\/a>Western immunoblotting<\/a><\/p>\n<\/a>\u00a0Coommassie blue staining of protein gels<\/a><\/p>\n\u00a0Site-directed mutagenesis<\/a><\/p>\n<\/a>PEI_Transfection_Bowdish_v2<\/a><\/p>\n\n
Cell Culture:<\/em><\/h2>\n
<\/a>\u00a0Counting cells with a hemocytometer and measuring viability with Trypan Blue.\u00a0<\/a><\/p>\n<\/a>Propagation & culture of THP-1 cells<\/a><\/p>\n\n
\u00a0Culturing RAW 264.7 Cells<\/a><\/p>\n\n
\u00a0Culturing HEK293\/HEK293T cells<\/a><\/p>\nCulturing, Maintaining and Seeding the nasopharyngeal cell line Detroit 562<\/a><\/p>\n\n
<\/a>Transformation of competent cells<\/a><\/p>\nCell signalling:<\/em><\/h2>\n
<\/a>NF-KB Luciferase Assay using HEK293T cells<\/a><\/p>\n\n
<\/a>Nf-KB-Secreted alkaline phosphatase using HEK293T cells<\/a><\/p>\n\n
<\/a>Nf-kB-secreted alkaline phosphatase protocol (version 2)<\/a><\/p>\n\n
<\/a>Growth and preparation of\u00a0Streptococcus pneumoniae<\/em><\/a><\/p>\n<\/a>Intranasal colonization of Streptococcus pneumoniae<\/em><\/a><\/p>\n\n
<\/a>CFSE labelling of S pneumoniae<\/em><\/a><\/p>\n\n
<\/a>NF-KB Luciferase Assay using HEK293T cells<\/a><\/p>\n\n
<\/a>Measurement of binding to intact bacteria<\/a><\/p>\n\n
Macrophage Killing Assay<\/a><\/p>\n\n
Macrophage Killing Assay<\/a>\u00a0version 1<\/p>\nMacrophage Killing Assay<\/a>\u00a0version 2<\/p>\n\n
<\/a>Pneumococcal Cell Wall Purification<\/a><\/p>\n\n
<\/a>Phenol-sulfuric acid Mannose detection assay<\/a>.<\/p>\n\n
<\/a>\u00a0Nasopharyngeal colonization, and measurement of gene expression and leukocyte recruitment in the nasopharynx.<\/div>\nMacrophage Isolation & Culture:<\/em><\/h2>\n
<\/a>Harvesting murine bone marrow<\/a>\u00a0from femurs and tibia<\/p>\nHuman monocyte derived macrophage culture<\/a><\/p>\nHarvesting murine\u00a0bone marrow from spines<\/a><\/p>\n\n
<\/a>Isolation & culture of murine bone marrow macrophages<\/a><\/p>\n<\/a>Elicitation and collection of peritoneal macrophages and neutrophils using Biogel<\/a><\/p>\nCalcein staining of<\/a>\u00a0macrophages<\/a><\/p>\nRNA collection and gene expression analysis:<\/em><\/h2>\n
<\/a>RNA extraction – lungs<\/a><\/p>\n<\/a>RNA extraction – nasopharynx<\/a><\/p>\n<\/a>RNA Extraction For RNAseq<\/a><\/p>\n\n
<\/a>RNA preparation for RNAseq<\/a> (RNA sequencing\/transciptomics)<\/p>\n\n
Metabolomics – Liquid Chromatography-Mass Spectrometry (LCMS)<\/em><\/h2>\n
<\/a>\u00a0Preparation of eukaryote and prokaryote cells for analysis of the endo- or exo-metabolome by LCMS<\/a><\/p>\nFlow cytometry based protocols<\/em><\/h1>\n
General<\/h2>\n
<\/a>An introduction to automated flow cytometry gating tools and their implementation<\/a><\/p>\n\n
<\/a>General Flow Cytometry protoco<\/a>l<\/p>\nBacterial flow cytometry<\/h2>\n
<\/a>Measurement of binding to intact bacteria<\/a><\/p>\n\n
Human flow cytometry<\/h2>\n
<\/a>\u00a0Human monocyte staining protocol<\/a><\/p>\n<\/a>\u00a0<\/a>Human lymphocyte staining protocol<\/a><\/p>\n<\/a>Human Treg (regulatory T cell) staining<\/a><\/p>\nMouse flow cytometry<\/h2>\n
<\/a>Flow cytometry protocol for staining Ly6Chigh and Ly6Clow monocytes<\/a><\/p>\n<\/a>\u00a0Cryopreservation with OCT<\/a><\/p>\n\n
\u00a0Microbiome – 16s sequencing<\/h2>\n
<\/a>Isolation of DNA from the murine nasopharynx<\/a><\/p>\n<\/a>Amplification of 16s rRNA for Illumina sequencing<\/a><\/p>\nScavenger receptor protocols<\/h2>\n