Question: How can I optimize my TotalSeq™ antibody labeling protocol?
Answer: There are a few critical workflow steps for staining with Cell Surface Proteins that should be highlighted for optimal assay performance.
(Note: These guidelines apply to both the TotalSeq panel and user-conjugated antibodies)
- Co-staining with antibodies for Feature Barcoding and FACS. Cells can be stained with antibodies for use with both flow sorting and Feature Barcoding simultaneously. To do this, we recommend preparing the pool separately, then adding them to the cell suspension at the same time. Please note that distinct antibody clones need to be selected for FACS and cell surface protein labeling if staining for the same cell surface protein for both workflows.
- Removal of antibody aggregates. Over time, antibodies tend to clump together and form aggregates. These aggregates will result in very high antibody UMI counts for the cells that they bind to, and they will appear in the downstream data as 'Antibodies in Barcodes with High UMI counts'. While these aggregates are removed from downstream analysis and therefore should not impact the rest of the data quality, they will make up a significant portion of your final library, resulting in wasted sequencing. In order to prevent the presence of aggregates, after making your antibody mix, centrifuge it at 14,000 rcf for 10 min at 4C. Transfer the supernatant (containing Antibody Mix) to a new tube and maintain at 4C. This step will leave the antibody aggregates at the bottom of the original tube.
- Thorough washing of your sample post-antibody staining. Thorough washing of your sample post-stain is critical to obtaining good data. Failure to fully remove the unbound antibody will result in high background, making it impossible to look for differential expression of the antibodies. We recommend a minimum of 4 washes, performed in 3.5 ml of wash buffer, in order to fully remove all the antibody that did not bind to cells. Additionally, we recommend performing the following steps after the first wash:
- Resuspend pellet in 100 ul PBS*
- Transfer suspension to a fresh tube
- Incubate at room temperature for 5 min*
- Add 3.5 ml of wash buffer and spin down for a second wash
- Perform an additional 2 washes
The goal of performing these steps is to leave unbound antibody sticking to the sides of the original tube, so this transfer step reduces the unbound antibody in the cell suspension.
*Please note that incubation in PBS and incubation at room temperature can be damaging to fragile cell types. If working with a fragile cell type, we recommend skipping the PBS resuspension and room temperature incubation. We still recommend performing a transfer to a fresh tube to reduce unbound antibody in the cell suspension. Please see our demonstrated protocol for more guidance on what constitutes a fragile cell type.
Please see the infographics below for guidelines on proper washing of cell suspensions stained with antibodies:
For more guidance on cell surface protein staining, please see our Demonstrated Protocol: Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols.
Products: Single Cell Gene Expression, Single Cell Immune Profiling