Protocols
Any time CometChip is performed on a new cell type, it is important to confirm that you are using appropriate parameters for analyzing your data set. This can be done by choosing a representative well image and analyzing it in a special “debug” mode in the Guicometanalyzer, as illustrated in this protocol.
The CometChip is a high-throughput version of the comet assay and is used to measure DNA damage. This protocol takes you through running the Guicometanalyzer code for analyzing CometChip images. The Guicometanalyzer code is freely available and can be found at the provided link: The Guicometanalyzer is available at https://github.com/audreyx0206/MalariaCometChip (Wood et al., 2010)
This protocol describes how to disaggregate whole colon tissue into a suspension of colonic crypts. This protocol was adapted from Samuel et al., Am J Physiol Cell Physiol 296: C296-C305, 2009. The sample image shows a suspension of crypts, with intact crypts outlined in red.
This protocol details how to fabricate the 96-well CometChip.
This protocol describes how to quantify any type of cellular foci, such as γH2AX stained images of tissue or cells, using the free NIH Image Processing and Analysis in Java software called ImageJ or Fiji.
A protocol for genotyping Aag-/- mice from ear punches or tail clippings. The Aag gene codes for alkyladenine DNA glycosylase, an enzyme that initiates repair of a variety of DNA lesions via the base excision repair pathway.
A protocol for genotyping AagTg+/- mice from ear punches or tail clippings. AagTg transgeneic animals have increased alkyladenine DNA glycosylase (Aag) activity via promoter-controlled expression of Aag cDNA.
A protocol for genotyping Mgmt-/- mice from ear punches or tail clippings. The Mgmt gene codes for O6-alkylguanine DNA alkyltransferase, which converts mutagenic O6-methylguanine lesions in DNA to guanine by demethylation.
This protocol describes the steps necessary to fluorescently detect proteins in paraffin-embedded tissue sections. After depariffinizing and rehydrating the tissues, antigens are exposed through incubation with boiling antigen retrieval buffer. Subsequently, sections are blocked with bovine serum albumin and probed with primary antibody overnight. After a series of washes, the sections are incubated with secondary antibody and detected with a fluorescent microscope.
This protocol describes how to make cell line lysates for downstream proteomic analysis by mass spectrometry. In this protocol, BaP is used as an example for treatment, but the process can be applied for other treatments.
These protocols describe the process to genotype for the RaDR transgene in mouse ear punches. RaDR mice were developed in the Engelward lab at MIT, and they allow detection of sequence rearrangement mutations arising from aberrant homologous recombination in situ. Please see the paper below for more information on RaDR.
The first protocol describes a quick tissue lysate procedure and the PCR recipe utilizes the commercial Platinum Supermix.
The second protocol in the document describes a tissue digestion procedure that yields purified DNA, and it provides a PCR recipe using individual ingredients that may be more cost-effective.
Engelward Lab protocol for creating microfeature molds from silicon wafers by lithographically patterning SU-8 photoresist.
This protocol describes how to covalently functionalize single-wall carbon nanotubes with iodonium salt and fabricate a CNT-based chemiresistive sensor for the detection of carbon monoxide.
Preparing conducting polymers by electrochemical polymerization for sensing applications.
This protocol describes how to disaggregate fresh tissues into single cell suspensions for analysis by flow cytometry. The protocol was developed for analyzing the proportion of EGFP+ cells in RaDR mouse tissues, but it can be adapted to any application requiring a suspension of single cells.
This protocol describes how to collect tissues for imaging EGFP foci in RaDR mice. Includes tips for clean excision and removing autofluorescent substances.
RNA extraction from tissue or low yield samples and preparation of RNA samples for RNA-sequencing (RNA-seq) using NuGEN's SPIA (Single Primer Isothermal Amplification) amplification technology.
Motivation
• DNA damage is associated with an increased risk of cancer, aging and disease
• Few methods are available for high throughput analysis of DNA damage
• This protocol is based upon the traditional comet assay wherein DNA migrates more
readily through a matrix when damaged
• 96 samples can be processed in parallel
• Throughput and sensitivity are increased compared to the traditional assay
This protocol describes how to immunofluorescence stain for Ki67, a marker of cellular proliferation, in paraffin-embedded tissue sections.
This protocol describes how to stain sections of formalin-fixed paraffin-embedded tissue for phosphorylated Ser139 on histone variant H2A.X (γH2AX). A primary antibody is used to identify and bind to γH2AX, and a secondary antibody conjugated to a fluorescent probe identifies and binds the primary antibody for imaging by fluorescence.
Description of how to prepare the cells and lanthanide solutions used for studying DNA damaging potential of these metals.
This protocol describes how to perform a Western blot using the BioRad Protean 3 Cell system, one of many available Western blot assemblies. The basic steps of this protocol can be adapted to any Western blot assembly, but we recommend reading the instructions for your system first.
