Sample recovery for Western blotting, electron microscopy and immunofluorescence microscopy after FASS

The isolation of specific subpopulations of synaptosomes by FASS aims at analyzing the biochemical composition of these specific synaptosomes by methods such as Western blotting, proteomics and immunofluorescence microscopy. Moreover, it is necessary to demonstrate the integrity of the synaptic particles after FASS using electron microscopy (EM). All of these methods require specific sample preparations, which had to be developed and optimized.

3.3.1 Vacuum filtration on polycarbonate filters is efficient in recovering synaptosomes for Western blotting and electron microscopy

During fluorescence activated sorting of synaptosomes, the particle solution is diluted by several orders of magnitude. In case of the FACSAria, set up with the 70 µm nozzle and sorting in “purity mode”, the average particle is sorted in a volume of roughly 2 nL of buffer.

This corresponds to 2 mL per million particles. Wolf et al. have estimated the protein content of 3 million sorted rat striatal synaptosomes to be in the range of 0.5 µg (Wolf and Kapatos, 1989a). Considering these estimates I was aiming at the isolation of 50 – 70 million particles in order to isolate 10 microgram of material. This would result in 100 to 140 mL of sorted sample, which have to be concentrated by a factor of 1000 in order to allow for analysis by Western blotting.

I therefore tested several strategies by concentrating a suspension of 10 µg (total protein) of B-fraction in 100 mL FACS-Sheath fluid (PBS) and determining the recovery by 1-D S1-DS PAGE and Coomassie staining (Summarized in Figure 13 A). With a recovery 56 % of protein, vacuum filtration through a 0.22 µm polycarbonate filter (Isopore, Millipore) proved to be the most effective method for recovering the diluted synaptosomes. The sorting of 50-70 million VGLUT1^VENUS synaptosomes takes several hours. In addition, the filtration of 10 µg (protein) of synaptosome takes 2-3 hours and requires the sorted material to be collected in a large container (100-140 mL). Plastic and glass surfaces can adsorb proteins and lipids unspecifically (Norde, 1992; Suelter and DeLuca, 1983). Therefore, also the sorted synaptosomes will be partly adsorbed to the surface of the collection device. I collaborated with the fine-mechanics workshop of the MPI of Experimental Medicine to design a collection

device that allows the vacuum filtration to occur in parallel to the sorting. We produced a water cooled, online vacuum-filtration device designed to fit the FACSAria, which allows the collection and vacuum filtration of 2 samples simultaneously (Figure 13A and Appendix 2).

In this way, the total time of the procedure, as well as the surface of the collection device were greatly reduced at the same time.

The Isopore filters were originally designed for the analysis of airborne contaminations by electron and light microscopy (Millipore homepage, particle concentration was also used to study the sorted material by EM (see 2.4.4 and 3.6.10)

Figure 13: Sample recovery after FASS

Several methodological developments were made to allow the efficient analysis of FASS samples. (A) Different strategies were tested to recover synaptosomal proteins from 100 mL of a diluted synaptosome suspension. The dilution used was similar to the dilution introduced by FASS. Filtration using 0.22 µm filters gave optimal results. (B) A custom-made vacuum-filtration device was designed to fit the FACSAria. During FASS, samples can be collected into either of two collection chambers. The wall of the collection chambers is lined by replaceable plastic vials, whose bottom was cut off and which can be replaced to reduce the

risk of cross-contamination between samples (5). The device can be attached to a circulating water cooling system (3) and a metal casing around the collection chamber ensures efficient cooling of the collected sample (6). The bottom of the collection chamber consists of a removable filter cassette (1). The filter cassettes can be taken apart and an Isopore filter can be placed on top of a perforated plastic disc (2). This perforated plastic disc supports the Isopore filter during the vacuum filtration. To drive the filtration, vacuum is applied below the filter cassette holder (4); a waste bottle connected in between the vacuum source and the filtration device collects the filtrate. After the sample has been filtered, the cassette holder can be removed and the filter can be processed for subsequent analyses. (C) Recovery of VGLUT1^VENUS synaptosomes after FASS for immunofluorescence staining. Up to 4 mL (roughly 2x10⁶ particles) of FASS sample can be centrifuged onto glass coverslips at up to 10.000xg in custom-made centrifuge adaptors for the Beckman JA7.2 swing-out rotor. Using serial dilutions of synaptosomes in PBS, a quantitative immunostaining of VGLUT1^VENUS

3.3.2 Centrifugation in custom adaptors allows quantitative recovery of diluted synaptosomes for immunofluorescence microscopy

synaptosomes using a GFP antibody revealed a linear recovery of particles even from heavily diluted synaptosome suspensions.

Even though the Isopore filters were originally designed for microscopy, fluorescence background prevented their effective use for the study of sorted particles by immunofluorescence microscopy (data not shown). Therefore alternative strategies were employed.

In order to quantitatively pellet all particles of a diluted synaptosome suspension onto gelatinized glass coverslips I collaborated with the MPI of Experimental Medicine fine mechanics workshop to design centrifuge adaptors that allow high-speed centrifugation. The adaptors were manufactured to hold four coverslips and fit into the Beckman JA 7.5 swing-out bucket rotor. For each coverslip a volume of 4 mL could be centrifuged. The JA 7.5 rotor has four buckets and therefore allows the simultaneous centrifugation of four adaptors holding a total of 16 coverslips at a maximum speed of 7,500 rpm (10,500 x g). The adaptors were manufactured from Delrin, which is suitable for centrifugation and resistant to salt, aldehydes and alcohol.

In order to test the quantitative recovery of synaptosomes from diluted suspensions, I made serial dilutions of a B-fraction with dilution factors of 1:1,000, 1:10,000 and 1:100,000.

3 mL of diluted synaptosomes were centrifuged at 10,500 x g for 20 min onto gelatinized glass coverslips. Subsequently, particles were stained for VGLUT1^VENUS using an anti-GFP antibody and epifluorescence images were quantified for number of VGLUT1^VENUS

Figure 13

positive particles per field ( C). Linear recovery of particles from different dilutions indicated that a representative fraction of particles was pelleted onto the coverslips. Additionally, when processing sorted synaptosomes for immunofluorescence microscopy by this method, the supernatant of the centrifugation was devoid of particles as analyzed by flow cytometry

(FACSAria, data not shown). This means that all particles that were detected by flow cytometry were quantitatively removed from the solution using this centrifugation protocol.

3.4 FASS in FSC-mode does not remove inhibitory synaptosomes