Frequently Asked Questions
MultiPro™ General FAQs
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 The MultiPro™ Human Fixed Cell Immune Profiling Antibody 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™ Human Fixed Cell Immune Profiling Antibody Cocktail?
A: The cocktail has been validated with resting and activated human PBMCs that were FC blocked and fixed with 4% PFA for 15 minutes 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 brands of antibody oligo conjugates?
A: You may be able to. Please contact us at genomics.inquiries@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 the MultiPro™ Human Fixed Cell Immune Profiling Cocktail (G900004).?
A: Yes. Please contact us at genomics.inquiries@ptglab.com if you need guidance on using MultiPro™ antibodies with other oligonucleotide-conjugated antibodies.
Q: Can you create custom cocktails for any single-cell or spatial platform?
A: Yes, we can create custom cocktails for most available single-cell or spatial platforms. Reach out at genomics.inquiries@ptglab.com for more information.
Q: What’s the maximum number of antibodies I can include in a custom cocktail?
A: We can create a custom cocktail of any size. There is no additional fee for creating a smaller cocktail, but creating a cocktail that includes more than 200 antibodies may require an additional fee.
Q: What is the turnaround time for a custom cocktail?
A: 6-8 weeks after receipt of all 3rd party antibodies. An additional 2-3 weeks will be required if custom validation services are required. Timelines will be discussed with you prior to quotation.
Q: Is there a basic validation performed on cocktails?
A: We offer screening of your custom cocktail against human PBMCs or mouse splenocytes, including using LPS and PMA stimulations.
Q: What kind of custom validation services do you offer?
A: We offer validation with cell types and conditions of your choosing.
Q: Do you provide custom conjugation services?
A: Yes, we do. Please reach out to genomics.inquiries@ptglab.com for inquiries.
Q: How much input material do you need for custom conjugations?
A: We require at least 0.5mg of each antibody to be conjugated. Typical yields are 10-20%. Yields are typically sufficient for 100+ staining reactions in single-cell applications.
Q: Can custom conjugated antibodies be included in a custom cocktail?
A: Yes, we can include custom conjugated antibodies in custom cocktails.
Q: Are your antibodies only reactive to human proteins?
A: Many of our antibodies are cross-reactive with many species, including mouse epitopes. Please reach out to genomics.inquiries@ptglab.com for information on specific antibody clones.
Q: In your example data, did you use protein transport inhibitors for cytokine staining?
A: In our example data, all stimulations were performed in the presence of commercially available protein transport inhibitors
Q: How long are MultiPro™ antibodies stable for?
A: 1 year after receipt of antibodies or cocktails
Q: How long will it take to get my MultiPro™ single antibodies?
A: In the USA, up to 1 week. In other regions, up to 2+ weeks.
Q: Where can I find the MultiPro™ Staining Protocol?
A: You can find the protocol here.
Q: Where can I find the 10x Genomics Cell Surface Protein Only Staining Protocol?
A: You can find the protocol here.
Sample Prep
Q: Your protocol for the MultiPro™ Human Fixed Cell Immune Profiling Antibody Cocktail (Cat# G900004) indicates that I should stain cells fixed with 4% 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 4% 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 4% 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 long-term storage
Q: Can I fix my cells for longer than 15 minutes?
A: Fixation for 15 minutes is an optimal fixation time for providing enough cellular structural integrity for permeabilization while also enabling successful staining with a wide range of antibodies. Fixation for longer periods is possible with minimal impact to RNA detection, however performance of some antibodies may be negatively impacted. For more information reach out to genomics.inquiries@ptglab.com
Cocktails
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 lyophilized cocktail?
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™ in the cocktail protocol to remove aggregates?
A: Antibodies in solution may form aggregates over time. Given the sensitivity of NGS based assays, even 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.
Q: Can I spike MultiPro™ single antibodies into the MultiPro™ Fixed Cell Immune Profiling Cocktail (G900004)?
A: Yes, single antibodies can be used alone or spiked into the MultiProTM Human Fixed Cell Immune Profiling Cocktail (G900004).
Staining
Q: How is cell permeabilization accomplished?
A: Cells are permeabilized with detergents NP-40 and Tween-20. Please see protocol for more details.
Q: Why do you stain after fixation? Doesn’t this negatively impact RNA integrity?
A: Fixation is critical for intracellular staining as exposure to detergents without fixation will lead to cell membrane disintegration. We have carefully validated our assay and determined that RNA performance is not hindered with our 15 min fixation.
Q: Can MultiPro™ oligo-conjugated antibodies be applied to single cell suspensions prepared from tissue?
A: Yes, MultiPro™ antibodies can be used to stain single cell suspensions derived from enzymatically digested tissue. However, enzymatic digestion may lead to reduction in the amount of target protein, requiring an increase in concentrations of antibodies used for staining. For more information reach out to genomics.inquiries@ptglab.com
Q: Can I use a single vial of cocktail to stain more than 1 million cells?
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 sample to mitigate the risk of low cell recovery or losing too many cells for loading the 10x Genomics Chromium Instrument.
Q: How much of MultiPro™ single antibodies should I use per staining reaction?
A: In general, we advise less than 0.5µg per 1 x 106 cells. This applies to single stains or if spiking into a cocktail. For more specific advice, contact a href="mailto:genomics.inquiries@ptglab.com">genomics.inquiries@ptglab.com
Q: Will the MultiPro™ Human Fixed Cell Immune Profiling Cocktail (G900004) work on other 10x Genomics assays besides Single Cell Gene Expression FLEX, a.k.a. Fixed RNA Profiling?
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.
Q: Can I use MultiPro™ antibodies with TotalSeq™ Antibodies?
A: This has not been validated, however the 5-CFLX oligos are similar to TotalSeqTM-C oligos. For more information, please reach out to genomics.inquiries@ptglab.com
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 MultiProTM libraries are distinct from parameters required for 10x Genomics Gene Expression Flex sequencing libraries. MultiProTM 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 MultiProTM sequencing parameters are:
Read 1:48 cycles
Index 1:10 cycles
Index 2:10 cycles
Read 2:50 cycles
Q: What sequencing depth is recommended for MultiPro™ antibodies?
A: We recommend adding 5K reads per cell on top of the sequencing depth being used for gene expression library sequencing. For example, if you are sequencing to a depth of 10K reads per cell for gene expression libraries, increase to 15K reads per cell when sequencing both gene expression and antibody libraries together. If sequencing antibody libraries separately, sequence to a depth of 5K reads per cell.
Analysis
Q: Do I need additional files for analysis?
A: The MultiPro™ Fixed Cell Immune Profiling Cocktail (G900004) has a own Feature Barcode Reference file, which is used when setting up Cell Ranger for analysis. This file 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.inquiries@ptglab.com