Human PBMCs Labelled With Extracellular and Intracellular Antibodies Using the Proteintech MultiPro™ Human Fixed Cell Immune Profiling Antibody Cocktail

Human PBMCs, obtained by 10x Genomics from AllCells, were placed in culture and rested overnight. The following day, the cells were subjected to one of three conditions:

1. Rest: Cells continued resting in culture.
2. PMA/Ionomycin stimulation: Cells were stimulated for 6 hours with PMA and ionomycin in the presence of protein transport inhibitors, brefeldin A and monensin, using the eBioscience™ Cell Stimulation Cocktail (plus protein transport inhibitors, Cat# 00-4975-93).
3. LPS stimulation: Cells were stimulated for 6 hours with 5 ng/mL LPS (InvivoGen LPS-B5 Ultrapure, Cat# tlrl-pb5lps) alongside protein transport inhibitors brefeldin A and monensin, using the eBioscience™ Protein Transport Inhibitor Cocktail (Cat# 00-4980-03).

After stimulation, cells were harvested and any cells attached to the flask were recovered with accutase treatment. The cells were then cryopreserved.

To perform antibody (Ab) labeling with extracellular and intracellular antibodies, cryovials from the three conditions were thawed. Cells were either fixed for 1 hour at room temperature (condition: Rested, no Ab) or Ab labeled (Conditions: Rested, PMA/Iono, LPS) with the MultiPro™ Human Fixed Cell Immune Profiling Antibody cocktail which contains 53 antibodies against cell surface and intracellular proteins plus five isotype control antibodies (Proteintech, Cat# G900004). Ab labeling was performed as described in the Intracellular Protein Labeling Protocol section of the Demonstrated Protocol Cell Surface & Intracellular Protein Labeling for GEM-X Flex Gene Expression (CG000781, Rev A). After Ab labeling, cells were then fixed for 1 hour at room temperature following Fixation of Cells & Nuclei for GEM-X Flex Gene Expression Demonstrated Protocol (CG000782).

Flex Gene and Protein Expression libraries were generated as described in the GEM-X Flex Gene Expression Reagent Kits for Multiplexed Samples with Feature Barcode technology for Protein using Barcode Oligo Capture User Guide (CG000789) and sequenced on an Illumina NovaSeq 6000 with approximately 39,000 paired end reads per cell for Gene Expression and 9,000 paired end reads/cell for Feature Barcode libraries.

Paired-end, dual indexing libraries were sequenced as follows:

• 28 cycles Read 1
• 10 cycles i7
• 10 cycles i5
• 90 cycles Read 2

Libraries were analyzed using the cellranger multi pipeline.

Number of cells called by condition:

• Rested, no Ab, 19,113 cells detected
• Rested, with Ab, 16,978 cells detected
• PMA/Iono induced, with Ab,19,200 cells detected
• LPS induced, with Ab, 18,298 cells detected

This dataset is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. 10x citation guidelines available here.