Critical step All solutions and buffers must be free of detergent and therefore should be prepared in plasticware, or in glassware thoroughly cleaned with ethanol. All reagents that can be stored in aliquots at −20 °C can be used for at least 6 months after preparation.
The medium consists of DMEM supplemented with 10% (vol/vol) horse serum, 5% (vol/vol) fetal calf serum, 4 mM glutamine and 100 U ml−1 each of penicillin and streptomycin. It can be stored at 4 °C and used within 1–2 weeks after preparation.
The solution consists of 320 mM sucrose and 5 mM HEPES, pH 7.4 (adjusted with NaOH). It can be stored at 4 °C and used within 1 week after preparation.
The solution consists of 250 mM sucrose and 3 mM imidazole, pH 7.4 (with HCl). It can be stored at 4 °C and used within 1 week after preparation.
The solution consists of 200 mM PMSF in ethanol. It can be stored at −20 °C and used within 1 week after preparation.
The solution consists of 1 mg ml−1 pepstatin A in DMSO. It can be stored in aliquots at −20 °C.
It comprises 10 mg ml−1 leupeptin hemisulfate in H2O. It can be stored in aliquots at −20 °C.
The solution is prepared by diluting 10 mg ml−1 aprotinin in H2O and can be stored in aliquots at −20 °C.
The solution contains 150-mM NaCl and 20-mM Na2HPO4 (disodium hydrogen phosphate), pH 7.4 (with HCl). It can be stored at room temperature (i.e., at 21 °C) for up to 2 months.
It should be prepared freshly for PNS preparation and consist of 5 mg ml−1 BSA in PBS.
The solution consists of 625 mM HEPES, 75 mM magnesium acetate and 10 mM DTT (pH 7.4, with KOH), and can be stored in aliquots at −20 °C.
The solution consists of 1 M potassium acetate in H2O and can be stored in aliquots at −20 °C.
The solution consists of 100 mM ATP in H2O (pH 7.4, with KOH) and can be stored in aliquots at −20 °C.
It comprises 800 mM creatine phosphate in H2O and is stored in aliquots at −20 °C.
It comprises 4 mg ml−1 creatine kinase (=3,200 U ml−1) in H2O and is stored in aliquots at −20 °C.
It consists of 250 mM glucose in H2O and can be stored in aliquots at −20 °C.
The internalization medium consists of 1 g D-glucose in one bottle (500 ml) OptiMEM and is stored in aliquots at −20 °C.
The setup consists of a Zeiss Axiovert 200M fluorescence microscope (Carl Zeiss AG) equipped with a ×100, 1.4 numerical aperture oil objective and a CCD camera with a 1,317 × 1,035 Kodak chip (pixel size 6.8 μm × 6.8 μm; Princeton Instruments Inc.). Blue fluorescence is detected using the excitation filter 350/50 D, the beamsplitter 400 DCLP and the emission filter 460/50 D. Alexa 488 fluorescence (absorption/emission maxima ~496/519 nm) is detected with the excitation filter 480/40 HQ, the beamsplitter 505 LP Q and the emission filter 527/30 HQ. DiI fluorescence (absorption/emission maxima ~554/571) is detected using the excitation filter 545/30 HQ, the beamsplitter 570 LP Q and the emission filter 610/75 HQ. Alexa 594 fluorescence (absorption/emission maxima ~590/617 nm) is detected using the excitation filter 560/55 HQ, the beamsplitter 595 LP Q and the emission filter 645/75 HQ. Alexa 647 fluorescence (absorption/emission maxima ~650/665 nm) is detected using the excitation filter 620/60 HQ, the beamsplitter 660 LP Q and the emission filter 700/75 HQ. All filters can be purchased from Chroma. For image acquisition, we use METAMORPH (Universal Imaging Corporation).
The homogenizer we use is manufactured by our in-house instrument shop and consists of a stainless steel shaft and a stainless steel ball (Spheric-Trafalgar Ltd). The clearance between the shaft and the ball has to be 20 μm. The ball homogenizer has been originally described in reference 14 in great detail. A commercially available variant of the ball homogenizer can be obtained from Isobiotech.
Critical Clearance has to be 20 μm for PC12 cells and may be different for other cells, e.g., 25 μm for BHK cells.
For imaging, we use a chamber that is manufactured by our in-house instrument shop, consisting of a circular metal base on which a plastic ring is screwed, thus holding the coverslip (see ref. 15 for a detailed description). We use this chamber, as it allows imaging in the presence of buffer. Any other design that allows imaging of coverslips is appropriate.