Leave tubes on ice and repeat Steps 13–15 for the next 6–12 plates of cells.
When all cells are collected, centrifuge all tubes at 250g for 5 min at 4 °C.
Discard the supernatant and resuspend the cell pellet in 10 ml of ice-cold PBS and transfer to a 15-ml plastic tube. Centrifuge again at 250g for 5 min at 4 °C.
Discard the supernatant and resuspend the cell pellet in 10 ml ice-cold internalization medium and centrifuge the cells as before (250g, 5 min, 4 °C). If more than one PNS label is needed (e.g., for docking/fusion assay) and two differently labeled PNS fractions are required, resuspend the cell pellet in 20 ml ice-cold internalization medium and divide into equal amounts in two 15-ml plastic tubes before centrifugation (250g, 5 min, 4 °C), for the separate PNSs.
Estimate the volume of the resulting cell pellet(s).
Critical step This is just an approximation but is necessary for calculating the amounts of fluorescent cargoes to be added. The volume should be in the range of 0.5–3 ml.
Remove supernatants and warm the pellets at 37 °C for 5 min in a water bath.
Add fluorescent cargoes in uptake medium to the cell pellets, as described in the options below, and incubate for 5 min at 37 °C. The medium should have the same volume as the estimated cell pellet(s) and should thus show cargo concentrations twice as high as finally desired (e.g., to label 1 ml cell pellet with 50 μg ml−1 fluorescent transferrin, 1 ml uptake medium with a concentration of 100 μg ml−1 transferrin is required). The cargo composition is dependent on whether the docking/fusion assay (A) or the sorting/budding assay (B) should be used as follows:
For dual labeling with transferrin and LDL: simultaneously incubate cells with 50 μg ml−1transferrin-Alexa 488 and 3 μg ml−1 LDL-DiI (prepared at 100 μg ml−1 transferrin and 6 μg ml−1 LDL in internalization medium).
Critical step LDL easily oxidizes, which affects the uptake efficiency. The exact concentration may need to be adjusted, with concentrations of up to 10 μg ml−1being needed after several weeks of storage. To delay the oxidation process, it is recommended to keep the LDL-DiI stock solution (Invitrogen) always sealed with parafilm.
For triple labeling with transferrin, LDL and cholera toxin, incubate cells with 50 μg ml−1transferrin-Alexa 488, 3 μg ml−1 LDL (both as above) and 3 μg ml−1 cholera toxin subunit B-Alexa 647 (i.e., a concentration of 6 μg ml−1 for the uptake medium). Note that any other combinations of labeled endocytotic cargoes can be used.
Incubate one set of cells with 500 μg ml−1 dextran Alexa 488, 10 kDa (prepared at 1 mg ml−1in internalization medium).
Incubate a second set of cells with 500 μg ml−1 dextran Alexa 594, 10 kDa (prepared at 1 mg ml−1 in internalization medium).
Docking/fusion assay
Sorting/budding assay
Stop the uptake reaction by chilling the cells on ice, centrifuge them at 250g for 5 min at 4 °C and remove the uptake medium.
Wash the cell pellet(s) three times with 10 ml ice-cold PBS-BSA; centrifuge after each wash at 250g for 5 min at 4 °C.
Prepare 30–50 ml homogenization buffer with protease inhibitors by adding PMSF, pepstatin A, leupeptin and aprotinin (1:1,000 (vol/vol) of the respective stock solutions).
Wash the cell pellet(s) once with ice-cold homogenization buffer and resuspend the pellet(s) in ice-cold homogenization buffer with protease inhibitors (Step 25).
Critical step Use no more than 4× the volume of the cell pellet or the PNSs will be too dilute.
Crack the cells with a ball homogenizer on ice. Use 1-ml disposable syringes for passing 1 ml of resuspended cells 10–20 times through the precooled ball homogenizer. Repeat the procedure, depending on the volume of cells.
Critical step Before cell cracking, wash the assembled ball homogenizer thoroughly with homogenization buffer (with protease inhibitors, Step 25) to remove air bubbles that would cause damage to the endosomes.
Transfer the cell homogenate into 1.5-ml tubes and centrifuge at 1,200g for 15 min at 4 °C. This will lead to a separation of the homogenate into two layers: the bottom fraction is the nuclear pellet and the upper fraction is the PNS.
Critical step It is sometimes difficult to see the separation of the two layers properly. Use a bright light source (lamp or bright window) to better illuminate the samples. If no band is visible, further dilute the samples with ice-cold homogenization buffer (with protease inhibitors, Step 25) and repeat the centrifugation process.
Remove the supernatant fractions into a new tube and determine the protein concentration of the PNS using a standard method16. The PNS concentration is usually between 10 and 16 mg ml−1.Troubleshooting
Prepare small aliquots (100–400 μl), snap-freeze in liquid nitrogen and store at −80 °C.Pause Point If stored at −80 °C, PNS fractions can be used for at least 12 months.
Points from here (point 31) up to and including point 39 are related to Preparation of docking/fusion or sorting/budding reactionsTiming: 2–4 hThaw aliquots of PNS (from Step 30), cytosol (from Step 8), DHM buffer, KAc buffer, ATP solution, CP solution, CK solution and homogenization buffer.