Summary of key steps in microparticle flow cytometry experimental workflow
| Purpose | Relevant figures | Experimental assays | Key notes |
|---|---|---|---|
| Determine detection sensitivity of cytometer | Fig. 1; Extended Data Fig. 1-1 | FSC vs fluorescence triggering with lipid dye (e.g., FM4-64 or other) PS and silica bead standards | A number of dyes are suitable for fluorescence triggering (Gray et al., 2015; Arraud et al., 2016; Kormelink et al., 2016; Stoner et al., 2016). Multiple studies, including ours, have demonstrated the increased sensitivity, accuracy, and reliability of fluorescence triggering (Nolan and Stoner, 2013; Arraud et al., 2016). FSC signal from bead standards (especially PS) does NOT accurately reflect the size of biological particles detected. Refractive index differences lead to size underestimation, the extent of which is highly cytometer dependent (Table 6). |
| Determine linear range of particle detection | Fig. 2; Extended Data Fig. 2-1 | Dilution series | Conducting a dilution series is critical to identify the range of linear detection on each cytometer. |
| Detection of coincidence and aggregation | Fig. 3; Extended Data Fig. 3-1 | Multicolor dye labeling | A number of dyes are suitable for multicolor dye labeling assays, as described in cellular “barcoding” assays (Krutzik and Nolan, 2006; Krutzik et al., 2011). |
| Distinguishing between coincidence and aggregation |
Fig. 4; Extended Data Fig. 4-1
Fig. 5 | (1) Dilution series with multicolor dye labeling (2) Sorting double-positive events from beads vs samples (3) Spin vs mix multicolor dye labeling | We found that mixtures of different color PS beads are a suitable model of coincidence. Double-positive sample events that do not decrease with dilution, and those that can be enriched by sorting, strongly suggest aggregates vs coincidence. This can be further supported by testing whether the frequency of double-positive events is increased by centrifugation. |
| Reduce aggregation |
Fig. 6; Extended Data Fig. 6-1
Fig. 7; Extended Data Fig. 7-1 | Use of nonionic buffers | As reported in previous microscopy studies (Choi et al., 2009; Daniel et al., 2012), we found that nonionic sample buffers tend to reduce aggregation of synaptosomes. This is not a complete solution to eliminating false double-positive and triple-positive events. |
| Identify suitable FSC ranges |
Fig. 6; Extended Data Fig. 6-1
Fig. 7; Extended Data Fig. 7-1 | Immunostaining and multicolor dye labeling | FSC of beads provides relative references for avoiding regions with high false double-positive rates |
| Assess false colocalization of antigens | Fig. 7; Extended Data Fig. 7-1 | Immunostaining of mutually exclusive antigens | Highly abundant antigens that should be mostly exclusive are best for these experiments. |