Protocols

These are protocols that are used in the Bowdish lab. We hope that these are in sufficient detail as to be helpful for you but if not please don’t be shy. If you’ve got questions or comments let us know and we’d be happy to help. Contact bowdish@mcmaster.ca or the individual who generated the protocol (email addresses on our “Group Members” page). We update these frequently in the hopes of providing the community with clear, concise and easy-to-reproduce protocols.

Bowdish lab specific documents:

 Yearly check-in  (undergraduate)

  • The purpose of the yearly check-in is to assess your progress and identify any roadblocks or obstacles to productivity. Usually done in January (1/2 way through a thesis project) but can be done whenever you would like some feedback on your performance.

Yearly check-in (graduate)

    • The purpose of the yearly check-in is to assess your progress and identify any roadblocks or obstacles to productivity. Usually done in January as this is a convenient time to reflect, but can be done whenever you would like some feedback on your performance

Guidelines for Laboratory notebooks and data

  • This document outlines expectations and requirments for how you maintain a laboratory notebook and manage your data during your stay in the Bowdish lab. All new members are required to read and follow these guidelines.

 Bowdish lab exit checklist

  • So you’re leaving the lab? Not without filling out this checklist!

General Protocols:

How to Write your first Manuscript

Cytokine ELISA 

  • This is an updated version of the original.

Methylene Green SDS Detection Assay

  • Is there SDS contamination in your reagent? Quantitate the amount of SDS using this assay based off of the traditional Methylene Blue assay.

Protein A Sepharose purification of Antibodies

  • This protocol is useful for purifying antibodies from hybridoma cultures and serum.

Molecular Biology:

Cloning

The PCR resource manual.

  • The how-to guide:qPCR from prime

The PCR resource manual.

  • The how-to guide:qPCR from primer design to testing those primers in preparation for quantitative PCR

Immunoprecipitation

  • This protocol demonstrates how to immunoprecipitate a protein from a cell lysate.

Co-immunoprecipitation

Western immunoblotting

 Coommassie blue staining of protein gels

 Site-directed mutagenesis

PEI_Transfection_Bowdish_v2

  • This is a cheap and effective protocol for transfecting HEK293T cells using PEI.  Note: In Jan 2013 we posted an updated version (version 2) with clarifications requested by users. We have another variant here. We have adapted this protocol for 24 well plates. To see the adapted version click Protocol for 24-Well Plates

Cell Culture:

 Counting cells with a hemocytometer and measuring viability with Trypan Blue. 

Propagation & culture of THP-1 cells

  •  Tips and tricks for growing this popular, but finicky, human monocyte-like cell line.

 Culturing RAW 264.7 Cells

  • The murine RAW 264.7 cell line is considered one of the best macrophage cell lines. This is an update of the original in we describe culture & propogation of the cells and include information on seeding density for studies of cytokine production.

 Culturing HEK293/HEK293T cells

Culturing, Maintaining and Seeding the nasopharyngeal cell line Detroit 562

  • This human derived nasopharyngeal carcinoma cell line maintains many of the properties of nasopharyngeal epithelial cells.

Basic Microbiology:

Transformation of competent cells

Cell signalling:

NF-KB Luciferase Assay using HEK293T cells

  • This protocol is used to measure activation of Nf-kb downstream of activation of TLRs or NLRs.

Nf-KB-Secreted alkaline phosphatase using HEK293T cells

  • This protocol is used to measure activation of Nf-kb downstream of activation of TLRs or NLRs.

Nf-kB-secreted alkaline phosphatase protocol (version 2)

  • Here is a slightly tweaked version of the NF-kB reporter assay used by some members of the lab.

Streptococcus pneumoniae:

Growth and preparation of Streptococcus pneumoniae

Intranasal colonization of Streptococcus pneumoniae

  • This is a detailed protocol for preparing Streptococcus pneumoniae and intranasal colonization of mice.

CFSE labelling of S pneumoniae

  • This protocol uses CFSE to label S. pneumoniae for flow cytometry or microscopy experiments.

NF-KB Luciferase Assay using HEK293T cells

  • This protocol can be used to test whether bacterial ligands induce activation of TLRs and NLRs.

Measurement of binding to intact bacteria

  • This protocol is used for “bacterial flow cytometry” (running bacteria through the flow cytometer rather than eukaroytic cells). We measure protein binding to the bacteria but it could easily be adapted to antibodies.

Macrophage Killing Assay

  • This is a standard kill curve protocol used to measure S. pneumoniae killing, can be adapted for any bacteria and any type of macrophage (e.g. human, murine, alveolar, bone marrow,etc).

Macrophage Killing Assay version 1

Macrophage Killing Assay version 2

  • How do you measure how well macrophages kill bacteria (or whether your treatment alters killing)? This is a standard kill curve protocol and a workhorse of the Bowdish lab. Specifically written to measure S. pneumoniae killing, can be adapted for any bacteria and any type of macrophage (e.g. human, murine, alveolar, bone marrow,etc).

Pneumococcal Cell Wall Purification

  • This protocol describes how to purify the pneumococcal cell wall. The purified preparation will not contain proteins but will contain the peptidoglycan layer and teichoic acids. It is updated from the original.

Phenol-sulfuric acid Mannose detection assay.

  • This assay is sensitive to mannose from 1 – 100 𝜇g but will also detect other carbohydrates. We use this assay to quantify of complex carbohydrates in the bacterial cell wall
 Nasopharyngeal colonization, and measurement of RNA and leukocyte recruitment.
For a detailed protocol, see our paper here…

Puchta A, Verschoor CP, Thurn T, Bowdish DM. Characterization of inflammatory responses during intranasal colonization with Streptococcus pneumoniae. J Vis Exp. 2014 Jan 17;(83):e50490. doi: 10.3791/50490.

Macrophage Isolation & Culture:

Harvesting murine bone marrow from femurs and tibia

Human monocyte derived macrophage culture

Harvesting murine bone marrow from spines

  • We have found that you get as much as 5x more bone marrow when collecting from the spines, as opposed to standard femur & tibias. 

Isolation & culture of murine bone marrow macrophages

Elicitation and collection of peritoneal macrophages and neutrophils using Biogel

Calcein staining of macrophages

RNA collection and gene expression analysis:

RNA extraction – lungs

RNA extraction – nasopharynx

RNA Extraction For RNAseq

  • This protocol is for isolating RNA from cell lines or tissues for RNA sequencing.

RNA preparation for RNAseq (RNA sequencing/transciptomics)

  • The protocol details the steps required fro processing (eukaryote) RNA for RNA sequencing experiments including ribosomal RNA depletion, quality control and quantification, cDNA preparation and labelling with adaptors. 

Metabolomics – Liquid Chromatography-Mass Spectrometry (LCMS)

 Preparation of eukaryote and prokaryote cells for analysis of the endo- or exo-metabolome by LCMS

Flow cytometry based protocols

Measurement of binding to intact bacteria

  • This protocol is used for “bacterial flow cytometry” (running bacteria through the flow cytometer rather than eukaroytic cells). We measure protein binding to the bacteria but it could easily be adapted to antibodies.

General Flow Cytometry protocol

 Human monocyte staining protocol

 Human lymphocyte staining protocol

Flow cytometry protocol for staining Ly6Chigh and Ly6Clow monocytes

An introduction to automated flow cytometry gating tools and their implementation

  • This review on automated gating tool for use in multi-parameter flow cytometry is published in Front. Immunol. 6:380. doi: 10.3389/fimmu.2015.00380

Tissue collection:

 Cryopreservation with OCT

  • Many of the tissues we preserve and share with our collaborators are collected in OCT. Here is a detailed protocol on collection and preservation. 

 Microbiome – 16s sequencing

Isolation of DNA from the murine nasopharynx

Amplification of 16s rRNA for Illumina sequencing

Scavenger receptor protocols

Class A Scavenger Receptor Blocking Protocol

  • This protocol uses the scavenger receptor inhibitors dextran sulphate and polyI (and control ligands) to block scavenger receptor binding and is used whether ligand binding occurs by the class A scavenger receptors.

Genotyping Scavenger Receptor A and MARCO (Msr1 & Marco) knockout mice

Measuring bacterial products in biological samples

Measuring LPS in plasma or serum

  • This protocol is used for measuring levels of LPS in mouse or human plasma or serum.

Generating polyclonal antibodies in rabbits

Immunization protocol for rabbits