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CODEX

Introduction

A major obstacle for fluorescence microscopy is the visualization of multiple proteins or other biomarkers in their native cell and tissue context. This obstacle is due to the unavoidable spectral overlap inherent in visualizing more than 5-6 fluorophores at a time. In contrast, highly multiplexed fluorescence imaging makes it possible to visualize 10s of proteins and biomarkers, within multiple cell and tissue structures on a single tissue section, thus providing meaningful context. This enables better understanding and modeling of biological processes in context to and relationship with the multitude of proteins and biomarkers that define specific cell phenotypes and tissue architecture. One such technology, pioneered and developed in Dr. Nolan’s lab at Stanford and now patented by Akoya Biosciences, is "CODEX". This technology makes use of a DNA barcode technology comprised of unique, complimentary, oligonucleotide sequences. One unique oligo sequence is conjugated to a primary antibody (against a protein or other biomarker target); the complimentary oligo sequence is conjugated to a fluorescent reporter dye. DNA base pairing, between the conjugated complimentary oligonucleotides, then enables highly specific detection. Iterative cycles of imaging 3 different conjugated fluorophores (reports) at a time, plus a DNA dye, results in detection of 10s of biomarkers in a single tissue slice.

To learn more about CODEX refer to Akoya’s website: https://www.akoyabio.com/codextm/technology

 

Contact information

Anum Khan anumkhan@stanford.edu

Jon Mulholland jwm@stanford.edu

Services/Pricing

Tissue prep and sectioning

Users are responsible for harvesting and generating tissue thin sections on the coverslip for CODEX experiments. You can also use the facilities below for tissue sectioning. CODEX can be done on both fresh frozen as well as FFPE tissues. The markers available for both tissue types varies. FFPE tissue also needs to be deparaffinized for antigen retrieval. Additional fees apply. Please consult with the CSIF staff listed before embarking on tissue processing for CODEX. NOTE: The tissue sections (~5 microns) must be on poly lysine coated coverslips (22 X 22mm).

Staining and CODEX runs / Prices

If the marker of interest is available through Akoya, users can provide thin tissue sections on poly-lysine coated coverslips to the facility. CSIF staff will perform the tissue staining and set up the CODEX run for imaging. Please contact CSIF staff for pricing if marker number is significantly different.

  Pricing:
First test run / coverslip. This is for test piloting, new untested tissue $500/6 validated markers
1 CODEX experiment/coverslip using 25 or less validated markers Base cost per tissue slice for 25 or less validated markers: $550 Cost per marker (@1:200) for 1- 25 validated markers: $14 For example: $900/per tissue slice with 25 validated markers (@1:200)
1 CODEX experiment/coverslip using 50 or less validated markers Base cost per tissue slice for 50 or less validated markers: $841.50 Cost per marker (@1:200) for 26-50 validated markers: $14 For example: $1,541.50/per tissue slice with 50 validated markers (@1:200)

Custom conjugation and validation

For markers of interest that are not available through Akoya, users can conjugate their own markers to a DNA barcode. Please contact CSIF Staff for details. Validation (not included) will need to be performed for the conjugated antibody.

  Pricing
Conjugation Marker conjugation: $660/antibody (~50 CODEX runs)

Analysis

CSIF has an analysis workstation in the facility specifically for CODEX image analysis. CSIF staff will walk you through the basic analysis pipeline.

Training

The service center is in the process of setting up our CODEX service. Currently we do not offer training and the CSIF staff scientist handles all the staining and imaging.

Validated Panels

https://www.akoyabio.com/codex/assay-reagents/

Literature

1. Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging.
https://doi-org.stanford.idm.oclc.org/10.1016/j.cell.2018.07.010

2. Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front.
https://doi.org/10.1016/j.cell.2020.07.005