CODEX – high parametric imaging of cells in a tissue context

Introduction/background

To reveal and understand the collective behaviors of cells in the tissue context, imaging technologies are ideal since they preserve the spatial information. Due to the complexity of cellular phenotypes, their interactions and diversity in function, simultaneous detection of many more markers than allowed with conventional fluorescence microscopes are desired.

Recently a new imaging platform called CODEX – Co-Detection by Indexing was invented, in the lab of Gary Nolan at Stanford. The CODEX technology allows for highly multiplexed analysis for up to 40 proteins using cyclic detection of DNA-indexed antibody panels (illustrated to the left). In 2018 the Cell Profiling Facility and the research group of Emma Lundberg at SciLifeLab, was able to purchase one of a few pre-market instruments and since the start of 2020 the Cell profiling Facility is now offering this technology as full service projects.

CODEX as a national service

The Facility performs full service projects with the CODEX platform starting from a tissue section obtained from the user. The entire sample preparation, panel design, image capture and processing is done by the application specialist in the facility. Upon completion the user receives raw images as well as processed images of all markers with single cell resolution and an fcs file with spatial coordinates and quantitative measurements of all protein markers. As part of your project we offer a standard cytometric analysis of your sample based on your scientific question and an introduction to the CODEX Multiplex Analysis Viewer (MAV), to allow you to further analyze your data. The CODEX MAV is an open source software, free to download as a plug in to Image J.

Samples

The CODEX platform is compatible with fresh frozen (FF) and formalin fixed paraffin embedded (FFPE) cells or tissue sections of up to 10 um thickness and xy- dimensions of 11×11 mm. The Cell Profiling Facility accept pre-cut tissue sections mounted onto glass slides compatible with the CODEX stage. For local users we can assist in sectioning as a fee for service cost. The sample can be one whole section or a few smaller sections. In cases of FFPE samples a tissue microarray (TMA) of 16-25 cores can be analyzed onto one slide. Upon sample arrival a standard quality control is performed before proceeding with sample preparation and staining. Only samples of approved quality will be processed further.

The CODEX platform allows detection of up to 40 protein markers within the same sample. We offer pre-conjugated antibodies validated for CODEX for FF and FFPE human samples and FF mouse tissue samples. The available protein targets for mouse and human samples can be found here: Read more about available antibodies and panels here: https://www.akoyabio.com/codextm/codex-antibodies

Antibodies

The CODEX System is a fluidics control instrument that integrates with an existing fluorescent microscope for the synchronization of the CODEX Assay and image collection.The overall procedure for a CODEX experiment follows the steps below:

Technology description

1. Tissue staining
   The tissue sample is mounted on a glass slide and stained for all targets in parallel. All antibodies are conjugated with a specific oligo barcode developed by the provide – Akoya Biosciences. The targets are then detected using a fluorescently labelled complimentary oligo.
2. CODEX and microscope set-up
   The stained tissue sample is mounted on the microscope stage and connected to the CODEX module. The CODEX device is set-up with all buffers and the complimentary oligos.
3. Image acquisition
   Acquisition is done automatically and targets are detected and imaged in cycles of three targets in each cycle. Dapi is used as a counter stain and compensate for drift during image acquisition. Currently, the set-up allows for 24 markers in total.
4. Image processing
   Images are deconvoluted and stitched.
5. Image analysis
   This includes cell segmentation and generation of a csv file summarizing signal intensities from the antibodies from each cycle.

For questions about the CODEX technology contact the Cell Profiling Facility cellpro.facility@scilifelab.se