Question: What was updated in the Demonstrated Protocol for Cell Surface Protein labeling CG000149 RevC?
Answer: The wash steps were optimized to improve assay performance by decreasing background from unbound antibodies, which increases the "Fraction Antibody Reads in Cells" in the final data.
Additional minor changes to the protocol were made to improve assay performance. Please refer to the list below of the specific changes between the old protocol (CG000149 RevB) and the updated protocol (CG000149 RevC), implemented in March 2021.
- RevB: Centrifuge cells
- RevC: First add PBS + 0.04% BSA for a total volume of 1 ml, then centrifuge cells
- Rationale: To streamline the workflow when working with multiple samples, the volume is brought up to 1 ml so that all samples can be centrifuged together
Cell centrifugation conditions
- RevB: 400 rcf, 5 min
- RevC: cell type-specific conditions: cell lines = 300 rcf 5 min; PBMCs = 400 rcf 5 min; Dissociated Tumor Cells = 150 rcf 10 min
- Rationale: The centrifugation conditions were optimized for different cell types
- RevB: Optional to include Dextran Sulfate for blocking
- RevC: No longer recommend Dextran Sulfate
- Rationale: Through additional in-house tests we found that Dextran Sulfate has variable effects on data quality and can either increase or decrease the specificity of the Cell Surface Protein labeling reaction, depending on the antibody and cell type. Therefore, to maximize compatibility across various cell types and antibodies, we no longer recommend blocking with Dextran Sulfate. These new recommendations to exclude Dextran Sulfate are consistent with Biolegend’s TotalSeq recommendations.
- RevB: Three washes in 1.5 mL tubes
- RevC: Four washes in 5 mL tubes. Includes a tube transfer step and 5 minute PBS incubation after wash 1.
- Rationale: The number of wash steps and the volume of the wash steps were increased to reduce background from unlabeled antibodies. The tube transfer step reduces background from antibodies adhered to the tube. The 5 minute incubation in PBS also reduces background, but this step can be skipped if the cells are fragile. Altogether, these updated wash conditions are expected to lead to improved application performance by increasing the “Fraction Antibody Reads in Cells” in the final data
Final cell resuspension
- RevB: resuspend in PBS + 0.04% BSA
- RevC: resuspend in PBS + 2% BSA
- Rationale: To decrease cell aggregation and minimize cell loss
Antibody centrifugation conditions for aggregate removal
- RevB: 14000 rcf, 10 min, room temperature
- RevC: 14000 rcf, 10 min, 4°C
- Rationale: The centrifugation temperature was updated to be consistent with BioLegend's recommendations for TotalSeq antibodies
Products: Single Cell Gene Expression, Single Cell Immune Profiling