Points from here (point 1) up to and including point 8 are related to Preparation of rat brain cytosolTiming: 4–5 hPrepare sucrose buffer with protease inhibitors (1 μg ml?1 pepstatin A (1:1,000 vol/vol of pepstatin A stock solution) and 0.2-mM PMSF (1:1000 vol/vol of PMSF stock solution)) for about 5 ml per rat brain.
Critical step Stir the buffer thoroughly while slowly adding PMSF stock solution, as PMSF might precipitate otherwise.
Kill 10–40 rats and remove their brains into a beaker with sucrose buffer (without protease inhibitors, kept on ice).
Caution Follow national and institutional guidelines for animal handling.
Wash brains 2–3 times with ice-cold sucrose buffer (without protease inhibitors) to remove most of the blood remaining in the solution.
Add 10 brains into the Glass/Teflon Potter Elvehjem Tissue Grinder, add about 5 ml ice-cold sucrose buffer (with protease inhibitors, from Step 1) per brain and homogenize brains by 10 strokes (one stroke consists of one up and down movement) at 900 r.p.m. with an overhead stirrer.
Caution Do not apply too much pressure during the first stroke, as the first homogenization of brains is difficult and should be performed slowly; the glass homogenizer might break otherwise.
Centrifuge homogenate at 3,000g (5,000 r.p.m. in a Sorvall SS-34 rotor) for 10 min at 4 °C. Transfer the supernatant into a fresh tube and discard the pellet.
Centrifuge the supernatant at 32,000g (16,500 r.p.m. in a Sorvall SS-34 rotor) for 15 min at 4 °C. Transfer the supernatant into a fresh tube and discard the pellet.
Centrifuge the supernatant at minimum 100,000g (we use 330,000g (90,000 r.p.m. in a Beckman 100.3 rotor)) for 30 min at 4 °C to obtain the cytosol in the supernatant.
Transfer the cytosol-containing supernatant into a fresh tube and divide into small aliquots (0.3–1 ml), determine the protein concentration using a standard assay16, snap-freeze the aliquots in liquid nitrogen and store them at ?80 °C. The protein concentration of the cytosol concentrations should be in the range between 6 and 10 mg ml?1.Pause Point If stored at ?80 °C, rat brain cytosol can be used for at least 12 months.
Points from here (point 9) up to and including point 10 are related to Culture of PC12 cellsTiming: 30 minSplit PC12 cells 1:6 into 6–30 plates (? 15 cm) using standard procedures for passaging PC12 cells (or any other cell line)17.
Critical step Dissociate the cells thoroughly, as otherwise clumps may remain that may lead to inefficient cargo uptake. We resuspend the cell pellet in 5 ml fresh PC12 culture medium and break cell aggregates by passing the cells 20 times though a 200-μl pipette tip attached to a 10-ml plastic pipette.
Allow cells to grow for several days until they reach 80% confluence.
Critical step Cells should reach a high degree of confluence; otherwise, it is difficult to detach them from the plates.
Points from here (point 11) up to and including point 30 are related to Preparation of postnuclear supernatantsTiming: 3–5 hPrepare PBS-BSA (freshly, 30 ml for each labeled PNS) and store on ice, together with thawed aliquots of internalization medium (15 ml per PNS), homogenization buffer (50 ml per PNS) and PBS (10 ml per PNS).
Prewarm trypsin/EDTA and PBS (37 °C), but store PC12 culture medium in the refrigerator.
Remove old medium from 6–12 plates of cells and wash each plate with 5 ml PBS.
Add 2 ml trypsin/EDTA per plate and incubate at room temperature with strong shaking.
Critical step Cells should be exposed to trypsin/EDTA only until they start detaching from the plates. Longer exposure to trypsin/EDTA might cause the degradation of receptors that are required for the uptake of fluorescent cargoes.
Stop the reaction with 5 ml cold PC12 medium per plate, collect the cells from the plates (using a 10-ml plastic pipette) and transfer them into 50-ml plastic tubes.
Critical step Cells need to be detached, which may require mechanical force such as repetitively squirting of the medium onto the plate. This procedure is easier when longer trypsin/EDTA incubations have been used (Step 14); it is more efficient in highly confluent cultures (Step 10).