Omni-Stainer + OT-2 automates the entire 5.5 hr (fresh-frozen tissue sections) or 7 hr (FFPE) CODEX (PhenoCyler™) staining process with ease and precision. With optional antibody screening mode!
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Akoya Biosciences' PhenoCycler™ system, formerly known as CODEX (CO-Detection by indEXing), is a revolutionary spatial biology technology that provides single-cell resolution for highly multiplexed analysis of 100+ biomarkers. This is achieved through the cyclic detection of DNA-indexed antibody panels. In the complex world of multicellular organisms, no cell exists in isolation. For instance, in the tumor microenvironment, 50% of cells are not tumor cells. Traditional histology attempts to image these intricate interactions one marker at a time, while genetic screening provides deep multidimensionality to resolve cell populations but loses the spatial context. PhenoCycler™ bridges this gap, offering both spatial context and the multidimensionality of omics approaches.
Parhelia Biosciences' Omni-Stainer™ system automates the sample preparation process of the PhenoCycler™ system, making it more efficient and reliable. The PhenoCycler™-Fusion, touted as "The Fastest Single-Cell Spatial Biology Solution," enables users to "map a million cells in 10 minutes." However, the sample preparation between tissue sections and the PhenoCycler fluidics devices remains a delicate staining process that takes at least 7 hours for FFPE samples or 5.5 hours for fresh frozen samples.
Previously, this staining protocol was a manual task, requiring meticulous care to avoid damaging valuable samples on extremely fragile glass coverslips. This complex procedure followed an extensive 100+ page manual, consuming many hours.
Now, with the Omni-Stainer™ system, the entire staining procedure is fully automated, allowing researchers to focus on the science, not the sample prep.
No more tiptoeing around fragile glass coverslips or thumbing through a 100+ page manual.
CODEX protocol
Reagents:
S1/[Hydration buffer + 1.6% PFA] (PBS with 0.5%BSA, EDTA) (Goltsev et al 2018, Cell)
Preblock: Staining Buffer (S2) with SS DNA and rabbit and mouse IgGs. Preblock is the solution that is used to dilate the antibodies into and to stain the section.
Plating buffer: R1 with ssDNA and HOechst
H2/CODEX Buffer
Protocol:
Slide or coverslip with FFPE or fresh frozen section.
Frozen section pretreatment
Pull out of -80 freezer dry/bring to RT on Drye Rite
Drop the section into RT acetone for 10 min
Dry the sample on a filter paper
Rehydrate (PBS, S1). Note – should not contain any detergents. When the protocol is done on the robot – the rehydration step is substituted by the mounting procedure (preparing flow cell). The slide or coverslip are dipped into the reservoir with hydration buffer (PBS/Hydration buffer) and are either positioned over the Support Pad or covered with a Cover Pad
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Starting this step the protocol is done robotically.
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Postfix (S1 with 1.6%PFA; PBS with 4% PFA) for 10min. Note – should not contain any detergents. This step is skipped if the FFPE sample is to be stained.
Wash/Equilibrate in Staining Buffer (S2) 30min.
Preblock (solutions from the row A of the 96 well plate are pipetted, called CODEX® Blocking Buffer in CODEX user Manual) for 15min at RT
Dispense the Stain (solutions from the second row of the 96 well plate are pipetted).
Optionally the protocol is paused here to allow the user to place the OMNI-stainer into the cold room or refrigerator
Unless the #8 is used – incubate for 3h at RT for fresh frozen or for 8 hours at RT for FFPE.
Wash 3 times with S2
Fix in Storage (High Salt) buffer with 4% PFA.
Wash in PBS
Apply RT methanol and incubate for 10 min
Wash with PBS
Postfixation with BS3: Dilute the F-reagent in PBS and pipet onto the section( PBS from the 12-trough is pipetted into the third row of the 96 well plate and this mix is dispensed into the section)
Wash with PBS
If the screening mode is False the protocol stops here with placing the section into the storage buffer.
From this point if the multicycle rendering process is to be done inside the Phenocycler Fusion – the slide should be processed according to the steps described for mounting of the stained slide for the Phenocycler FUsion protocol.
If the multicycle rendering process is to be done inside the original CODEX robot the coverslip has to be placed section up into the microscope stage of the original CODEX robot and further imaged according to the instructions.
If the screening mode is True
The section is “stripped” (iterative washes with R1(screening 20%DMSO in H2) [Screening buffer (16mL 1x CODEX buffer mixed with 4mL DMSO)]and R2(stripping 80%DMSO in H2) buffers)
The section is rendered: detector oligos (in plating buffer) from the fourth row of the 96 well plate are pipetted onto the section and incubated for 10 min
The section is washed in R1
The section is stored in the Storage buffer.
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That is the end of the robotic protocol
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To image the slide or coverslip – wash in CODEX buffer, optionally pre-stain with DAPI/Hoechst for 1 min (these steps could be done manually in the OMNI-stainer or on in the 6-well plate) and either mount in CODEX buffer with nail polish or in any permanent Fluoromount system followed by imaging ()
