Intracellular Staining

TotalSeq™-B and -C Reagents for Intracellular and Cell Surface Protein Staining

Intracellular proteins contribute to a host of cellular functions including cell growth, gene regulation, chemotaxis, and immunomodulation. Measuring both intracellular and surface proteins at the single-cell level can give researchers a comprehensive understanding of the cell and the molecular processes that underlie cellular function.

The ability to detect cell surface proteins with gene expression in single-cell analysis has been well established using CITE-Seq. However, detecting intracellular proteins while preserving RNA quality for gene expression profiling has proven challenging, due largely to the conditions utilized to access the intracellular compartment of cells which can have deleterious effects on RNA quality.

With advancements in single-cell RNA technology and workflow optimization, it is now possible to observe cell surface, cytoplasmic, and nuclear proteins using TotalSeq antibodies. To preserve antigen integrity and epitope accessibility in different cellular compartments -cytoplasmic and nuclear- we have developed two distinct protocols. Each protocol uses a specialized buffer system optimized to ensure robust protein staining while maintaining a high-quality RNA signature when paired with the 10x Genomics Flex Gene Expression assay.

Why add intracellular targets to your single-cell experiment?

Improve cell characterization by identifying functional molecules.
Add depth to cell state identification by understanding signaling pathways.
Discover unique and new phenotypes within seemingly homogeneous cell populations.

Our protocols for detection of cytoplasmic or nuclear proteins can be found below.

TotalSeq™ Format Compatibility

View all TotalSeq-B and -C Intracellular Antibodies

Assay Compatibility

TotalSeq Format Compatibility	TotalSeq-B Surface and Intracellular Antibodies	TotalSeq-C Surface and Intracellular Antibodies
Single Cell Assay Compatibility	10x Genomics Chromium Single Cell Flex Gene Expression
BioLegend TotalSeq Intracellular Staining Protocol	TotalSeq™-B or -C Cell Surface and Intracellular Cytoplasmic Protein Staining with 10x Chromium Single Cell Gene Expression Flex
	TotalSeq™-B or -C Cell Surface and Nuclear Protein Staining with 10x Chromium Single Cell Flex Gene Expression
Instrument Compatibility	Chromium X Series
Description	Probe based whole transcriptome gene expression, compatible with fresh or fixed samples
Additional Cellular Features	Surface and Intracellular Proteins
Multiplexing Compatibility	TotalSeq-B Hashtags	TotalSeq-C Hashtags

*Use of BioLegend’s intracellular staining protocols and cell hashing reagents with 10x Genomics Chromium Single Cell Gene Expression Flex assay is not officially supported by 10x Genomics. For additional information regarding use of TotalSeq-B and -C intracellular antibodies or TotalSeq Hashtags please contact BioLegend Technical Services.

Workflows

TotalSeq™-B or -C Cell Surface and Cytoplasmic Protein Staining with 10x Chromium Single Cell Gene Expression Flex

TotalSeq™-B or -C Cell Surface and Nuclear Protein Staining with 10x Chromium Single Cell Gene Expression Flex

Required User Guides and Support

TotalSeq Format	TotalSeq-B	TotalSeq-C
BioLegend TotalSeq™ Intracellular Staining Protocols	TotalSeq™-B or -C Cell Surface and Intracellular Cytoplasmic Protein Staining with 10x Chromium Single Cell Gene Expression Flex
	TotalSeq™-B or -C Cell Surface and Nuclear Protein Staining with 10x Chromium Single Cell Flex Gene Expression
10x User Guides	Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling
	Chromium Fixed RNA Profiling Reagent Kits for Singleplexed Samples With Feature Barcode technology for Protein*	Flex Gene Expression User Guides**
10x General Support	Flex Gene Expression Support

Important:

*TotalSeq-B products are only compatible with 10x Genomics Chromium Fixed RNA Profiling Reagent Kits for Singleplexed Samples With Feature Barcode technology for Protein.

**TotalSeq-C products can be used with GEM-X v1 and v2 Flex Gene Expression Reagent Kits for Singleplex or Multiplex samples with Feature Barcode technology for Protein Expression, and Next GEM Fixed RNA Profiling Reagent Kits for Singleplexed or Multiplexed Samples with Feature Barcode technology for Protein using Barcode Oligo.

For additional support in choosing the correct Flex Gene Expression user guide to pair with our TotalSeq products please contact BioLegend Technical Services.

TotalSeq Reagents

TotalSeq-B: The capture sequence is compatible with the Capture Sequence 1, or Partial Capture Sequence 1 of the barcoded Gel Bead primers used with the 10x Genomics 3' Universal Gene Expression or Flex Gene Expression assays, respectively. Use of TotalSeq-B antibodies with these assays is fully supported by 10x Genomics.

TotalSeq-C: The capture sequence is compatible with the Template Switch Oligo Capture Sequence, or the Partial Capture Sequence 1 of the barcoded Gel Bead primers used with the 10x Genomics 5' Universal Gene Expression or Flex Gene Expression assays, respectively. The use of TotalSeq-C antibodies with these assays is fully supported by 10x Genomics.

TotalSeq Intracellular Antibodies	TotalSeq-B Intracellular	TotalSeq-C Intracellular
Cocktails	TotalSeq-B Cocktails	TotalSeq-C Cocktails
Hashtags	TotalSeq-B Hashtags	TotalSeq-C Hashtags
All Products	TotalSeq-B	TotalSeq-C