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Multiplexing-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

Cedric Espenel
espenel@stanford.edu

More information

Akoya’s Validated antibody Panels: CODEX Antibody Product List: Human FFPE

Akoya PN

CODEX Antibodies

Hu FFPE

4150020

Pan-Cytokeratin-BX019 (AE-1/AE-3)—Alexa FluorTM 488-RX019

FFPE

4150024

Actin-BX010 (2F1-1)—Alexa FluorTM 488-RX010

FFPE

4250012

CD8-BX026 (C8/144B)—Atto 550-RX026

FFPE

4250021

E-cadherin-BX014 (4A2C7)—Atto 550-RX014

FFPE

4250023

CD45RO-BX017 (UCHL1)—Atto 550-RX017

FFPE

4350021

Histone H3 Phospho (Ser28)-BX030 (HTA28)—Cy5-RX030

FFPE

4450020

Pan-Cytokeratin-BX019 (AE-1/AE-3)—Alexa FluorTM 750-RX019

FFPE

4450024

Actin-BX010 (2F1-1)—Alexa FluorTM 750-RX010

FFPE

4150017

CD31-BX001 (EP3095)—Alexa FluorTM 488-RX001

FFPE

4150018

CD20-BX007 (L26)—Alexa FluorTM 488-RX007

FFPE

4250002

CD44-BX005 (IM7)—Atto 550-RX005

FFPE

4250004

Podoplanin-BX023 (NC-08)—Atto 550-RX023

FFPE

4250019

Ki67-BX047 (B56)—Atto 550-RX047

FFPE

4350001

CD107a-BX006 (H4A3)—Cy5-RX006

FFPE

4350002

CD45-BX021 (2D1)—Cy5-RX021

FFPE

4350018

CD4-BX003 (EPR6855)—Cy5-RX003

FFPE

4350019

CD68-BX015 (KP1)—Cy5-RX015

FFPE

4350020

CD11c-BX024 (118/A5)—Cy5-RX024

FFPE

4450017

CD31-BX001 (EP3095)—Alexa FluorTM 750-RX001

FFPE

4450018

CD20-BX007 (L26)—Alexa FluorTM 750-RX007

FFPE

Akoya’s Validated antibody Panels: CODEX Antibody Product List: Human Fresh Frozen

Akoya PN

CODEX Antibodies

Hu FF

4150016

CD9-BX028 (HI9a)—Alexa FluorTM 488-RX028

FF-human

4150020

Pan-Cytokeratin-BX019 (AE-1/AE-3)—Alexa FluorTM 488-RX019

FF-human

4150021

CD90-BX022 (5E10)—Alexa FluorTM 488-RX022

FF-human

4150022

CD7-BX025 (CD7-6B7)—Alexa FluorTM 488-RX025

FF-human

4250020

CD34-BX035 (561)—Atto 550-RX035

FF-human

4250022

CD69-BX041 (FN50)—Atto 550-RX041

FF-human

4250023

CD45RO-BX017 (UCHL1)—Atto 550-RX017

FF-human

4350017

CD89-BX030 (A59)—Cy5-RX030

FF-human

4450016

CD9-BX028 (HI9a)—Alexa FluorTM 750-RX028

FF-human

4450020

Pan-Cytokeratin-BX019 (AE-1/AE-3)—Alexa FluorTM 750-RX019

FF-human

4450021

CD90-BX022 (5E10)—Alexa FluorTM 750-RX022

FF-human

4450022

CD7-BX025 (CD7-6B7)—Alexa FluorTM 750-RX025

FF-human

4150003

CD45-BX001 (HI30)—Alexa FluorTM 488-RX001

FF-human

4150004

CD8-BX004 (SK1)—Alexa FluorTM 488-RX004

FF-human

4150007

CD38-BX007 (HB-7)—Alexa FluorTM 488-RX007

FF-human

4150008

CD138-BX010 (MI15)—Alexa FluorTM 488-RX010

FF-human

4150009

CD21-BX013 (Bu32)—Alexa FluorTM 488-RX013

FF-human

4150010

CD63-BX013 (H5C6)—Alexa FluorTM 488-RX013

FF-human

4250004

Podoplanin-BX023 (NC-08)—Atto 550-RX023

FF-human

4250005

CD2-BX002 (RPA-2.10)—Atto 550-RX002

FF-human

4250006

HLA-DR-BX026 (L243)—Atto 550-RX026

FF-human

4250008

CD104-BX005 (58XB4)—Atto 550-RX005

FF-human

4250009

CD31-BX032 (WM59)—Atto 550-RX032

FF-human

4250010

CD279-BX014 (EH12.2H7)—Atto 550-RX014

FF-human

4250013

CD278-BX017 (C398.4A)—Atto 550-RX017

FF-human

4250019

Ki67-BX047 (B56)—Atto 550-RX047

FF-human

4350003

CD19-BX003 (HIB19)—Cy5-RX003

FF-human

4350007

CD49f-BX033 (GoH3)—Cy5-RX033

FF-human

4350008

CD3-BX015 (UCHT1)—Cy5-RX015

FF-human

4350010

CD4-BX021 (SK3)—Cy5-RX021

FF-human

4350011

CD5-BX024 (UCHT2)—Cy5-RX024

FF-human

4350012

CD11c-BX027 (S-HCL-3)—Cy5-RX027

FF-human

4450003

CD45-BX001 (HI30)—Alexa FluorTM 750-RX001

FF-human

4450004

CD8-BX004 (SK1)—Alexa FluorTM 750-RX004

FF-human

4450007

CD38-BX007 (HB-7)—Alexa FluorTM 750-RX007

FF-human

4450008

CD138-BX010 (MI15)—Alexa FluorTM 750-RX010

FF-human

4450009

CD21-BX013 (Bu32)—Alexa FluorTM 750-RX013

FF-human

4450010

CD63-BX013 (H5C6)—Alexa FluorTM 750-RX013

FF-human

Akoya’s Validated antibody Panels: CODEX Antibody Product List: Mouse Fresh Frozen

Akoya PN

CODEX Antibodies

Mu FF

4150015

CD11b-BX025 (M1/70)—Alexa FluorTM 488-RX025

FF-Mouse

4250017

CD8a-BX029 (53-6.7)—Atto 550-RX029

FF-Mouse

4350016

CD71-BX027 (RI7217)—Cy5-RX027

FF-Mouse

4450015

CD11b-BX025 (M1/70)—Alexa FluorTM 750-RX025

FF-Mouse

4250007

CD5-BX017 (53-7.3)—Atto 550-RX017

FF-Mouse

4150001

CD90.2-BX001 (30-H12)—Alexa FluorTM 488-RX001

FF-Mouse

4150002

CD45-BX007 (30-F11)—Alexa FluorTM 488-RX007

FF-Mouse

4150005

Ter119-BX004 (TER-119)—Alexa FluorTM 488-RX004

FF-Mouse

4150006

CD45R/B220-BX010 (Ra3-6B2)—Alexa FluorTM 488-RX010

FF-Mouse

4150011

IgM-BX013 (RMM-1)—Alexa FluorTM 488-RX013

FF-Mouse

4150012

IgD-BX016 (11-26c.2a)—Alexa FluorTM 488-RX016

FF-Mouse

4150013

CD38-BX019 (90)—Alexa FluorTM 488-RX019

FF-Mouse

4150014

CD24-BX022 (M1/69)—Alexa FluorTM 488-RX022

FF-Mouse

4250001

CD31-BX002 (MEC13.3)—Atto 550-RX002

FF-Mouse

4250002

CD44-BX005 (IM7)—Atto 550-RX005

FF-Mouse

4250003

MHC II-BX014 (M5/114.15.2)—Atto 550-RX014

FF-Mouse

4250014

CD19-BX020 (6D5)—Atto 550-RX020

FF-Mouse

4250015

CD21/35-BX023 (7E9)—Atto 550-RX023

FF-Mouse

4250016

CD4-BX026 (RM4-5)—Atto 550-RX026

FF-Mouse

4250019

Ki67-BX047 (B56)—Atto 550-RX047

FF-Mouse

4350004

CD279-BX006 (RMP1-30)—Cy5-RX006

FF-Mouse

4350005

CD169-BX015 (3D6.112)—Cy5-RX015

FF-mouse

4350006

TCRb-BX003 (H57-597 )—Cy5-RX003

FF-mouse

4350007

CD49f-BX033 (GoH3)—Cy5-RX033

FF-Mouse

4350013

CD11c-BX030 (N418)—Cy5-RX030

FF-Mouse

4350014

CD3-BX021 (17A2)—Cy5-RX021

FF-Mouse

4350015

Ly6g-BX024 (1A8)—Cy5-RX024

FF-Mouse

4450001

CD90.2-BX001 (30-H12)—Alexa FluorTM 750-RX001

FF-Mouse

4450002

CD45-BX007 (30-F11)—Alexa FluorTM 750-RX007

FF-Mouse

4450005

Ter119-BX004 (TER-119)—Alexa FluorTM 750-RX004

FF-Mouse

4450006

CD45R/B220-BX010 (Ra3-6B2)—Alexa FluorTM 750-RX010

FF-Mouse

4450011

IgM-BX013 (RMM-1)—Alexa FluorTM 750-RX013

FF-Mouse

4450012

IgD-BX016 (11-26c.2a)—Alexa FluorTM 750-RX016

FF-Mouse

4450013

CD38-BX019 (90)—Alexa FluorTM 750-RX019

FF-Mouse

4450014

CD24-BX022 (M1/69)—Alexa FluorTM 750-RX022

FF-Mouse

 

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 $350/6 validated markers
1 CODEX experiment/coverslip using 25 or less validated markers Base cost per tissue slice for 25 or less validated markers: $500 Cost per marker (@1:200) for 1- 25 validated markers: $12 For example: $800/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: $765 Cost per marker (@1:200) for 26-50 validated markers: $12 For example: $1,365/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.

Pricing
Conjugation Marker conjugation and validation: $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. If the analysis entails anything more project specific or complex, CSIF scientist (Cedric Espenel) can work with you to develop a customized algorithm.

Training

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