Critical step Be careful to thaw PNS and cytosol slowly on ice to avoid unwanted protein degradation or breaking of organelles. This might require up to 30 min, depending on the size of the aliquots.
Critical step Store aliquots of PNS in the dark to avoid bleaching of the fluorescent cargoes.
Sonicate multifluorescent TetraSpeck beads for 2–5 min in the bath sonicator and prepare a 1:1,000 (vol/vol) bead stock solution in PBS (2-μl beads in 2 ml PBS). This can be kept at 4 °C for up to 2 weeks.
Wash 18-mm glass coverslips in 100% ethanol and place them into 12-well tissue culture plates.
Dilute bead stock solution (from Step 32) 1:100 (vol/vol) in PBS to obtain the final bead solution and pipette 1 ml of it into each well. Remove the air bubbles that might be present between the plate and the coverslip to avoid coverslips floating on centrifugation.
Centrifuge plates at 5,880g for 45 min at 4 °C (we use 5,460 r.p.m. in a Heraeus Multifuge); this fixes the beads onto the coverslips. Do not remove the solution after centrifugation.
Prepare cytosol mix in the following order on ice, depending on whether an ATP-regenerating system (A) or an ATP-depleting system (B) is required, according to whether a positive control (in presence of ATP) or a negative control (in absence of ATP) is desired:
Centrifuge 5 μl hexokinase (1,500 U ml−1, supplied as ethanol precipitate) at 16,000g for 2 min at 4 °C.
Remove the supernatant and resuspend the hexokinase pellet in 5 μl glucose solution (250 mM).
Cytosol mix (volumes correspond to one reaction of 50 μl; scale up accordingly): 100 μg cytosol (usually 10–17 μl, depending on the concentration), 0.9 μl DHM buffer, 2.25 μl KAc buffer and 5 μl hexokinase-glucose buffer (see above).
The final concentration of the reaction therefore corresponds to 100 μg cytosol, 11.25 mM HEPES, 1.35 mM magnesium acetate, 0.18 mM DTT, 45 mM potassium acetate, 7.5 U hexokinase and 25 mM glucose.
Prepare cytosol mix (volumes correspond to one reaction of 50 μl, scale up accordingly): 100 μg cytosol (usually 10–17 μl, depending on the concentration), 0.9 μl DHM buffer, 2.25 μl KAc buffer, 1.67 μl ATP solution, 1.67 μl CP solution and 1.67 μl CK solution.
The final concentration of the reaction corresponds therefore to 100 μg cytosol, 11.25 mM HEPES, 1.35 mM magnesium acetate, 0.18 mM DTT, 45 mM potassium acetate, 3.3 mM ATP, 26.7 mM creatine phosphate and 6.7 μg creatine kinase (=5.3 U).
ATP-regenerating system
ATP-depleting system
Prepare docking/fusion (A) or sorting/budding (B) reactions in 200 μl polycarbonate centrifuge tubes on ice.
Critical step Pipette quickly to avoid warming of the solutions. Certain processes (e.g., docking or aggregation) might take place even during short pipetting times.
Add cytosol mix (from Step 36, usually between 18 and 25 μl).
Add chemicals, inhibitory/modulating proteins, peptides, antibodies (optional; see Experimental design for further information).
Add 200 μg PNS containing transferrin-Alexa 488 and LDL-DiI (or other double- or triple-labeled PNS) (usually between 10 and 15 μl).
Adjust to 50 μl with homogenization buffer.
Add cytosol mix (from Step 36, usually between 18 and 25 μl).
Add chemicals, inhibitory/modulating proteins, peptides, antibodies (optional; see Experimental design for further information).
Add 100 μg PNS containing dextran-Alexa 488 and 100 μg PNS containing dextran-Alexa 594 (usually between 5 and 8 μl each).
Adjust to 50 μl with homogenization buffer.
Docking/fusion reaction
Sorting/budding reaction
Close reaction tubes with a lid and incubate reactions for 45 min at 37 °C with a slow agitation (speed: 20 shakes per minute in the GFL 1086 water bath). Leave controls on ice for 45 min.
Critical step Samples should be kept in the dark to avoid bleaching the fluorescent dyes.
Place the samples on ice for some minutes to cool, pipette 7 μl of each reaction onto the bead-containing solution on the coverslips (from Step 35) and centrifuge the 12-well plates at 5,880g (5,460 r.p.m. in a Heraeus Multifuge) for 45 min at 4°C.
Points from here (point 40) up to and including point 43 are related to Imaging of the samplesTiming: 0.5–6 hPlace one coverslip into the open imaging chamber for 18-mm coverslips, cover it with PBS and focus the samples.
Acquire images in the channels of the respective label (Alexa 488, DiI, Alexa 594, Alexa 647, depending on the PNS fractions that are used) and in one additional channel in which only fluorescent beads can be visualized (e.g., blue) with an epifluorescence microscope (see EQUIPMENT SETUP).
Critical step Be careful with bleedthrough between the different channels and adjust the exposure times to minimize it.Troubleshooting