Use of gradient purified synaptosomes (B) results in faster sorting and higher purity compared to crude synaptosomes

(P2)

Previously published studies on flow-cytometry and fluorescence activated sorting of synaptosomes employed different subcellular fractions as starting material. While Wolf and colleagues used gradient purified synaptosomes (B) (Wolf and Kapatos, 1989a; Wolf and Kapatos, 1989b), Gylys and colleagues preferred the use of P2 fractions (Gylys et al., 2000;

Gylys et al., 2004a). The preference for using the P2 or the B fraction depends on two key parameters. Firstly, the initial degree of purification of fluorescent synaptosomes might influence the speed of sorting as well as the yield and the purity of sorted synaptosomes.

Secondly, the time passing between homogenization of the brain tissue and the sorting step might influence the sample quality. I therefore compared the FASS method with crude synaptosomes (P2; Figure 10) and gradient purified synaptosomes (B; Figure 11) as starting material.

During the preparation of synaptosomes, synaptic particles are already pre-enriched in the post-nuclear pellet P2. In my protocol the preparation of the P2 requires a combined 25 min of centrifugation time (Figure 6). From the P2, synaptosomes are further enriched by

discontinuous sucrose-density gradient centrifugation. The density-gradient purification adds more than one hour of centrifugation time to the protocol (2.3.1, Figure 6).

When analyzing subcellular fractions by flow cytometry, I analyzed the following parameters: FSC- forward angle light scattering, SSC - 90°-angle light scattering also called side scatter and VGLUT1^VENUS

Appendix 1

fluorescence. The signals measured for each of these parameters consists of three components, namely the height (H), the width (W) and the area (A) of the signals (for further explanations see ).

The VGLUT1^VENUS P2 preparation contained 38.3 % particles above wild-type background fluorescence (Figure 10 A). In order to avoid the analysis of aggregated particles and particles that are coinciding at the time of detection I selected particles of low SSC-W and FSC-W in the “small singles” gate (see Figure 10) for gate definition). After this gating step 7 % of all VGLUT1^VENUS

After sorting of the fluorescent particles of the P2, the sorted particles can be re-analyzed using the FACSAria (Figure 10 D). In order to control for unspecific effects of the sorting step on the sample, I also sorted and re-analyzed all P2-particles non-selectively (Figure 10 C). The re-analysis showed that sorting the fluorescent particles results in a more homogenous and smaller particle size and complexity distribution as indicated by the forward- and side-scatter analysis (FSC-A/SSC-A in Figure 10 D). In the fluorescent sample 35.9 % of particles were within the “ungated fluorescence” gate (Figure 10 D). The “ungated fluorescence” gate includes all particles that are above the wild-type background fluorescence irrespective of their light-scatter (FSC/SSC) signals. When all particles were sorted and re-analyzed, only 11.7 % of particles were in the “ungated fluorescent” gate (Figure 10 C). Thus, sorting of the fluorescent events of the P2-fraction resulted in a 3.1-fold enrichment of fluorescent particles, but only reached a total of 35.9 % fluorescent particles after FASS.

P2-particles had a fluorescence signal above the wild-type background and were selected using the “fluorescent” gate (Figure 10 B). For simplicity, the term ‘fluorescent particles’ will refer to the fluorescent events after the gating step that avoids aggregates.

Figure 10: FASS of VGLUT1^VENUS

Flow analysis and sorting of crude synaptosomes from wild type and VGLUT1 crude Synaptosomes (P2)

VENUS

crude synaptosomes (P2). (A) Analysis of wild type and VGLUT1^VENUS crude synaptosomes before sorting. The left plots show the analysis of light scattering in forward angles (FCS) and at 90°

angle (SSC), which represent the relative size and internal complexity of the particles, respectively. The central plots display the relative size (FSC) and the fluorescence intensity in the VENUS channel. On the right the fluorescence intensity distribution of the P2 samples of wild type and VGLUT1^VENUS P2 particles is represented in a histogram. (B) Gating strategy to preferentially select single particles above wild-type background fluorescence. In a first step a gate was drawn to include particles of small SSC-width and FSC-width in order to preferentially select single events. Next, the particles in this gate were analyzed for their fluorescence in the VENUS channel, a gate was drawn to include particles above wild-type background fluorescence. (C) Flow cytometric re-analysis of sorted P2 particles from VGLUT1^VENUS mice. In this experiment all P2 particles were sorted and reanalyzed. (D) Flow cytometric re-analysis of sorted, fluorescent P2 particles from VGLUT1^VENUS mice. In this experiment particles, which fell into the fluorescent gate (see VE-P2 in (B)), were sorted and re-analyzed.

When gradient-purified VGLUT1^VENUS

Figure 11

synaptosomes were used as starting material for FASS, 56 % of all particles were above wild-type background fluorescence and 14.7 % of particles sample fell into the single particle fluorescent gate ( AB). The re-analysis of sorted material revealed that, when all particles were sorted and re-analyzed 45.8 % of the events were above wild-type background fluorescence (Figure 11 C). In contrast, when the fluorescent particles of the B-fraction were sorted and re-analyzed 78.4 % of the particles were detected as fluorescent (Figure 11). Thus, sorting from gradient-purified VGLUT1^VENUS

Additionally, the B-fraction contained twice more (14.7 %) single, fluorescent events than the P2-fraction (7.0 %). This increase in the fraction of positive events, resulted in twice faster sort rates during fluorescence activated sorting in the FACSAria, resulting in a sort time of roughly 60 min for 10 million fluorescent particles when sorting from B.

synaptosomes resulted in a 1.7-fold enrichment of fluorescent particles and reached a total of 78.4 % fluorescent particles after FASS.

In summary, the preparation of gradient-purified synaptosomes is much more time consuming, but results in a 2.2-fold higher purity of VGLUT1^VENUS synaptosomes as analyzed by flow cytometry. In addition, the higher fraction of positive particles in the B-fraction allows for faster sorting, which decreased the total time for isolation of larger numbers of VGLUT1^VENUS synaptosomes by FASS.

Figure 11: FASS on gradient purified Synaptosomes (B)

Flow analysis and sorting of gradient purified synaptosomes (B) of wild type and VGLUT1^VENUS knock-in mice. (A) Analysis of wild type (top) and VGLUT1^VENUS (bottom) synaptosomes before sorting. The left plots show the analysis of light scattering in forward angles (FSC) and at 90° angle (SSC), which represent the relative size and internal complexity of the particles, respectively. The central plots display the relative size (FSC) and the fluorescence intensity in the VENUS channel. On the right the fluorescence intensity distribution of wild type and VGLUT1^VENUS B particles is represented in a histogram. (B) Gating strategy to select preferentially single particles above wild-type background fluorescence. In order to preferentially select single events a gate was drawn to include particles of small SSC-width and FSC-width. Next the particles in this gate were analyzed for their fluorescence in the VENUS channel and a gate was drawn to include particles above wild-type background fluorescence. (C) Flow cytometric re-analysis of sorted P2 particles from VGLUT1^VENUS mice. In this experiment all P2 particles were sorted and reanalyzed. (D) Flow cytometric re-analysis of sorted, fluorescent P2 particles from VGLUT1^VENUS

3.2 Aggregates within the B-fraction can be reduced by passage