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Can Xenium In Situ Gene Expression data be generated from PBMCs?

Question: Can Xenium In Situ Gene Expression data be generated from PBMCs?

 

Answer: Below is a summary of our learnings and considerations from working with PBMCs on Xenium. Methods for adherent cells can be found in: Can cells be adhered to Xenium slides to obtain gene expression data?

 

In general, modifications to documentation (Demonstrated Protocols and User Guide) provided here are unsupported. We cannot guarantee assay performance even when implementing the guidance listed in this article and therefore, customers should proceed at their own risk. 10x Genomics Support will not be able to provide additional information beyond this article, details from internal experiments, or replacement reagents in the event of assay failure (cell detachment, low number of transcripts per cell).

 

From limited testing, we have found that PBMCs yielded good data on the Xenium platform. This protocol outlines methods for fixation and adherence of PBMCs to Xenium slides for downstream processing with either Xenium v1 or Xenium Prime.

 

! These methods are to be used in place of demonstrated protocols CG000579 and CG000581, for preparation and fixation/permeabilization of fresh frozen tissue. No deparaffinization, decrosslinking, or sectioning is required for this protocol. In addition, sterility is not required.

 

Reagents:

  • 1X PBS, 5ml
  • 1X Complete Media, 10ml
  • PN-2000517 Fixation Buffer with the Conc. Fix & Perm Buffer 
    • From Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (PN-1000414)
  • Formaldehyde 37% (Fisher)

Xenium v1 specific reagents and consumables

  • PN-3000941 - 2 Xenium Slides, part of the Slide and Sample Prep Reagent Kit (PN-1000460)
    • Customers can also purchase the Xenium Slide Kit (PN-1000659, 4 rxn, or PN 1000660, 16 rxn) separately
  • PN-1000487 - 2 Xenium Cassettes and 2 Xenium Lids, part of the Xenium Decoding Consumables
    • Customers can also purchase Xenium Cassettes (PN-1000566) separately

Xenium Prime specific reagents and consumables

  • PN3000941 - 2 Xenium Slides, Xenium Prime Sample Preparation Reagents with Slides (PN-1000741)
    • Customers can also purchase the Xenium Slide Kit (PN-1000659, 4 rxn, or PN 1000660, 16 rxn) separately
  • PN-1000723 - 2 Xenium Cassettes v2 and 2 Xenium Lids, part of the Xenium Prime Decoding Consumables (PN-1000726)

 

Methods:

Buffer Dilution

Prepare Fixation Buffer according to CG000478

  1. Freshly dilute concentrated Fix & Perm buffer (PN-2000517) from 10x to 1x with nucleus free water and Formaldehyde from 37% stock to 4%.
  2. Prepare sufficient Fixation Buffer to resuspend cells. For two slides worth of buffer (1000 ul) add  791.9 ul of water, 108.1 ul of 37% Formaldehyde, and 100 ul of Conc. Fix & Perm Buffer.

 

PBMC Preparation and Fixation

  1. Collect cells in complete media and perform cell count.
  2. Wash and resuspend cells in Fix and Perm Buffer at 1e6 per ml.
    • Any size conical can be used, so long as it contains enough volume for all necessary cells.
      • Each slide requires 500ul, 5e5 cells total.
    • Incubate cells in Fix and Perm Buffer at room temperature (RT) for 1hr.

 

Cell Adhesion

  1. Assemble Xenium slides in cassettes.
  2. Spin cells and wash with 1ml PBS to remove excess Fixation and Permeabilization Media.
    • For detailed instructions on cell thawing and resuspension please refer to CG000447. 
  3. Resuspend cells at 1e6 per ml in PBS.
  4. Add 500ul (5e5 total cells) of cell suspension to each slide and cover with cassette lid.
    • Incubate at RT for 20 mins.
  5. Remove excess PBS, and gently wash Xenium slide with 500ul PBS 3x at RT.
  6. Add 500ul of PBS to each slide.

 

Xenium v1 Assay Workflow

Proceed to protocol CG000582 and continue with Probe Hybridization with desired probe panel. There are no modifications to this User Guide for PBMCs.

 

Xenium Prime Assay Workflow

Proceed to protocol CG000760 and the Xenium Prime Human Pan Tissue & Pathways Panel - 5K (PN-1000724). There are no modifications to this User Guide for PBMCs.

 

Product: Xenium In Situ Gene Expression, Xenium Prime In Situ Gene Expression