Question: How can I reduce background PE signal for the BEAM-Ab Negative Control Assembly?
Answer: When performing the BEAM-Ab pre-screening experiment (step 1 of protocol CG000595), if high background is observed in the PE channel for the Negative Control Assembly, the suggestions below may improve results.
1) Use a biotinylated off-target antigen to load and assign as the Negative Control Assembly
Our protocol CG000595 uses an empty/unloaded assembly as the Negative Control Assembly (prepared from PBS + BEAM Conjugate). However, after further testing, we found that high background in the PE channel may be observed when using this Negative Control Assembly to label B cells isolated using bead-based negative selection kits.
We found that the high background could be mitigated by preparing the Negative Control Assembly with a biotinylated off-target antigen, instead of using an empty/unloaded Negative Control Assembly (see Figure 1 below). For human samples, we recommend biotinylated human serum albumin (HSA-H82E3 from Acro Biosystems). For mouse samples, we recommend biotinylated mouse serum albumin (MSA-M82E4 from Acro Biosystems). This guidance has now been added to Revision B of protocol CG000595.
The smallest offering size from Acro Biosystems is 25 ug. This can be used to generate 125 ul of a 0.2 ug/ul stock solution, which is sufficient for 9 BEAM-Ab Assemblies (need ~13 ul per Assembly, based on the molecular mass of ~70 kDa).
2) Perform the quenching step during pre-screening.
The main purpose of the quenching step in the BEAM-Ab protocol is to reduce the risk of antigen swapping when multiple BEAM-Ab Assemblies are pooled together. Given that we do not pool multiple Assemblies together in the pre-screening protocol, the original version of our pre-screening protocol (CG000595 Rev A) did not include a quenching step. However, after further testing, we found that quenching can help reduce background PE signal in the Negative Control Assembly (see Figure 1 below). Therefore, we now recommend including the quenching step during pre-screening. This guidance has now been added to Revision B of protocol CG000595.
3) Avoid BSA-containing buffers
We recommend using PBS + 2% FBS for the BEAM labeling and wash steps. We do not recommend using alternative buffers. In particular, we do not recommend using BSA-containing buffers. Through recent BSA spike-in experiments, we found that BSA can increase the background PE signal in the BEAM-Ab workflow.
Residual BSA from bead-based enrichment kits may contribute to increased background. The Miltenyi Biotech MACS B cell isolation kit uses PBS + 0.5% BSA as the wash buffer. If bead-based enrichment is desired, selecting a kit without BSA may be preferable. Our current recommendation is StemCell EasySep B cell isolation kit, which uses PBS + 2% FBS as the wash buffer. However, it is still possible to use the MACS kit, as long as a biotinylated off-target antigen is used for the Negative Control (see Figure 1 below).
If bead-based enrichment kits or alternative buffers are used during sample preparation, we recommend performing a wash in PBS + 2% FBS prior to BEAM labeling. The following steps are recommended:
- Spin cells at 300 rcf for 5 min at 4C
- Resuspend in 5 ml chilled PBS + 2% FBS
- Perform cell count and viability check
- Dispense cells into a new 15 ml tube for BEAM labeling
- Spin cells at 300 rcf for 5 min at 4C
- Resuspend in 90 ul chilled PBS + 2% FBS
- Proceed with BEAM labeling (CG000595)
4) Perform sufficient wash steps after labeling
Ensure that sufficient wash steps are performed after BEAM labeling, to reduce background from non-specific binding. We recommend performing three wash steps. For each wash step, add 3.5 ml fresh, cold PBS + 2% FBS and pipette mix 5x.
Figure 1: Performing quenching and using biotinylated-HSA to prepare the Negative Control Assembly reduces background PE signal during BEAM-Ab prescreening. (A) Cells isolated using MACS B Cell Enrichment kit and labeled with an empty/unloaded Negative Control BEAM-Ab Assembly display high PE background, with 23.7% of cells in the PE positive gate. (B) Quenching the Assembly modestly reduces PE background, leading to 19.2% of cells in the PE positive gate. (C) Using biotinylated Human Serum Albumin (HSA) to prepare the Negative Control Assembly dramatically reduces PE background, leading to less than 1% of cells in the PE positive gate.
Products: Single Cell Immune Profiling, Barcode Enabled Antigen Mapping (BEAM)