Frequently Asked Questions


General

Q: Where is the protocol?

A: The protocol can be found here.

 

Q: What are MultiPro antibodies and how do they work with single-cell workflows?

A: MultiPro antibodies are oligonucleotide conjugated antibodies that enable the quantification of intracellular and cell surface proteins when performing single-cell RNAseq (scRNAseq)

 

Q: What single-cell workflows does your cocktail work with?

A: The MultiPro Human Fixed Cell Immune Profiling Antibody Cocktail is designed to work with only the 10x Genomics Chromium Single Cell Gene Expression Flex with Feature Barcoding and Sample Multiplexing workflow.

 

Q: What sample types are compatible with the MultiPro Fixed Cell Immune Profiling Cocktail?

A: The panel has been validated for resting and activated human PBMCs that were FC blocked and fixed with 4% PFA prior to staining. The cocktail targets human proteins and should be compatible with any human or human-derived cells but may require additional optimization.

 

Q: My protein of interest is not targeted by the cocktail, what are my options?

A: We are constantly evaluating and screening antibodies from the Proteintech antibody portfolio.­ If you need an antibody that is not included in our cocktail, please contact us at genomics.inquiries@ptglab.com

 

Q: Can I mix MultiPro with other brand antibody oligo conjugates?

A :Please contact us at genomics.support@ptglab.com if you need guidance on using MultiPro antibodies with other oligonucleotide-conjugated antibodies.

 

Q: Can I label my own antibody and spike it into your cocktail?

A: Please contact us at genomics.support@ptglab.com if you need guidance on using MultiPro antibodies with other oligonucleotide-conjugated antibodies.

 

Q: Will you provide single antibodies?

A: Currently, we do not provide single antibodies.

 

Q: Can I order custom cocktails?  

A: For inquiries about custom cocktails or antibodies, please reach out to us at genomics.inquiries@ptglab.com

 

Sample Prep

Q: Your protocol indicates that I should stain cells fixed with 2% PFA. Can I stain cells that have been fixed in some other way?

A: The MultiPro Human Fixed Cell Immune Profiling Antibody Cocktail (Cat# G900004) has only been validated with cells fixed with 2% PFA for 15 minutes.  If your study design requires different fixation conditions, please reach out to us at genomics.inquiries@ptglab.com

 

Q: Can I place my cells in long-term storage after the first 2% PFA fix?

A: We recommend proceeding until after “Sample Fixation” in the demonstrated protocol, “Fixation of Cells & Nuclei for Chromium Fixed RNA (GC000478)”. Follow guidelines for short- or long-term storage.

 

Cocktail preparation

Q: Why are my MultiPro cocktails lyophilized?

A: Lyophilization provides several benefits, including long-term stability and reduction of shipping/storage footprint.

 

Q: How do I store my cocktails?

A: Unused cocktail tubes should be stored at 2 - 8 C in the sealed mylar pouch in which they came. The desiccant packet should be kept in the same pouch.

 

Q: Why are there specific steps in the MultiPro protocol to remove aggregates?

A: Antibodies in solution may form aggregates over time. Given the sensitivity of NGS based assays, even very small amounts of antibody aggregates can have negative impacts on NGS data quality by reducing the efficiency of NGS via consumption of sequencing depth and reduction of dynamic range for on target signals. As a precautionary measure, any aggregates that may be present in an antibody cocktail can be effectively removed via centrifugation of the cocktail prior to cell staining.

 

Staining

Q: Can I use a single vial of cocktail for multiple samples?

A: Upon reconstitution, the cocktail is pre-titrated for staining up to one million cells per sample. Dilution of these cocktails to stain larger numbers of cells could impact product performance and data quality. Whenever possible, we highly recommend starting with 1 million cells per samples to mitigate the risk of low cell recovery or losing too many cells for loading the 10x Genomics Chromium Instrument.

 

Q:  Will the cocktail work on 10x Genomic Assays that take viable cells as input?

A: The cocktail is designed to be compatible with 10x Genomics Chromium Single Cell Gene Expression Flex product, which requires fixed cells as an input.

 

Sequencing

Q: What sequencing run configuration should I use? Illumina? Element?

A: After library construction, 10x Genomics Chromium Single Cell Gene Expression Flex libraries are ready for Illumina sequencing. Libraries are also compatible with the Element AVITI Adept workflow. The sequencing parameters for MultiPro are distinct from those required for 10x Genomics Gene Expression Flex sequencing libraries. MultiPro sequencing parameters are also distinct from other commercially available oligonucleotide conjugated antibodies. Please be mindful of this when setting up your sequencing run. The required MultiPro sequencing parameters are:
Read 1:48 cycles
Index 1:10 cycles
Index 2:10 cycles
Read 2:50 cycles

 

Q: What sequencing depth should I target for protein sequencing libraries?

A: We recommend a minimum of 10,000 reads per cell for protein libraries

 

Analysis

Q: Do I need additional files for analysis?

A: Each cocktail has its own Feature Barcode Reference file, which is used when setting up Cell Ranger for analysis. These files can be found here.

 

Q: How do I set up Cell Ranger for analysis?

A: MultiPro data requires 7.2.0 and above for analysis. Currently, Cell Ranger 7.2.0 is only available as a stand-alone package requiring local install. For further assistance in setting up Cell Ranger, please contact us at genomics.support@ptglab.com