During all the experiment, time and temperature are critical. All the steps have to be performed at +4°C if not otherwise stated. Pre-cool all the centrifuges and rotors.
An overview of the protocol is shown in Figure 1.
Preparation of zebrafish embryos extracts
1- Collect embryos at the one-cell stage in a 35 mm Petri dish with E3 medium (100 embryos per condition at least).
2- Place the embryos at the expected developmental stage into a pre-chilled dounce glass and add 1 ml of ice-cold MB buffer.
3- Homogenize for 10 times in ice with a loose-fitting pestle by slowly stroking the pestle up and down until all embryo’s chorions are removed. This step also permits to disrupt the yolk sac of the embryos.
4- Let the homogenate settle on ice for 10 minutes.
5- Transfer the homogenate into a pre-chilled 1.5 ml microcentrifuge tube and centrifuge at 300 g for 5 min.
6- An optional additional centrifugation step can be added. Re-spin the supernatant at 300 g for 5 min to pellet any cells left.
7- Resuspend pellet containing the cells with 1 ml of cold-MB buffer.
Isolation of mitochondria
8- Disrupt the cells with a 1 mL syringe and a 26 G x 2/3 needle 50 times on ice.
9- Homogenization can be checked by placing 2 µl on a microscope slide and visualizing cell lysis by phase-contrast microscopy. Few intact cells must be visible, if it is not the case re-disrupt homogenate until all the cells are lysed.
10- Centrifuge cell lysate twice at 1,500 g for 10 min to eliminate nuclei.
11- Transfer the supernatant containing mitochondria, cytosol and microsomes into a new pre-chilled 1.5 ml centrifuge tube.
12- Spin the supernatant twice at 10,600 g for 10 min to pellet mitochondria (labeled as mito-I).
13- Remove and store supernatant on ice (labeled as ER-I) for endoplasmic reticulum membranes isolation.
14- Gently resuspend the mito-I pellet with 1 ml of cold MB buffer.
15- Centrifuge again at 10,600 g for 10 min.
16- Discard the supernatant and save the pellet as mitochondria.
17- Gently resuspend mitochondria in 50 µl of cold MB buffer.
18- Determine the protein concentration of the sample (Bradford).
19- Store at -20°C for further investigations (western blot) if not used immediately.
Isolation of ER membranes
20- Place the ER-I supernatant (from step 13) into a pre-chilled ultracentrifuge tube and equilibrate the rotor carefully.
21- Spin at 100,000 g for 1 hour at +4°C.
22- The pellet is not visible so carefully discard the supernatant and gently resuspend the pellet containing ER membrane in 50 µl of cold MB buffer.
23- The amount of microsome proteins is determined (Bradford).
24- Store at -20°C for further investigations (western blot) if not used immediately.
Mitochondrial calcium uptake
25- Wash twice the mitochondrial pellet mito-I (from step 12) with 1 ml of fresh cold KCL medium.
26- Centrifuge 10 min at 10,600 g at +4°C.
27- Determine the amount of mitochondrial proteins (Bradford).
28- Transfer 75 µg of proteins into a new 1.5 ml centrifuge tube and adjust volume to 1 ml with KCL medium.
29- Centrifuge 10 min at 10,600 g at +4°C.
30- Gently resuspend the pellet with 180 µl of KCL medium containing 1 μM of the cytosolic calcium probe OregonGreen 5N.
31- Transfer all the volume in a well of a black 96-well plate with flat and transparent bottom.
32- OregonGreen 5N fluorescence intensity is measured using a Mithras LB 940 multimode microplate reader with excitation at 485 nm and emission at 510 nm.
33- Fluorescence intensity is measured every 2 s for 5 min.
34- After 30 s measurement, 20 µl of KCL buffer supplemented with 200 µM CaCl2 is injected (final concentration of CaCl2 20 µM).
35- The decrease of fluorescence intensity following the peak induced by CaCl2 injection is representative of the mitochondrial calcium uptake.