TITLE: Isolation of mitochondria from Diplonema papillatum
AUTHORS: Matus Valach

[STEPS]
?. Inoculate 50 mL of OSS growth medium supplemented with yeast extract (0.05%) and chloramphenicol (40 mg/L) with an axenic stock of Diplonema cells. Cultivate at 16 °C with occasional shaking (e.g. 4× per day) to cell density approx. 5×106 – 107 per mL. This corresponds approximately to the late exponential phase and usually takes 3-6 days, if starting from a frozen stock of cells.Cultivation at 16 °C seems optimal, but any temperature in the range from 15 °C to 20 °C works.Note that Diplonema papillatum will grow well in the range from 4 °C to 26 °C; however, the cultivation times will vary (e.g., slower growth at low temperatures) and the N2 pressure required for optimal lysis (see step #9) will need to be optimized (e.g., we noticed that cultivation in the cold generally results in higher pressures needed to disrupt the cells, which nevertheless makes avoiding mitochondrial leakage trickier).
?. Prepare 4 L of fresh OSS (or OSSYE) medium by adding horse serum (1% final) and, optionally, yeast extract (0.05% final) to the sterile artificial see water. Transfer the inoculum into large volume of the fresh medium (10 mL per 1 L, i.e., 100× dilution) and distribute the fresh culture (4 L) into large sterile flasks/containers with high Surface:Height ratio (see the guidelines).
?. Cultivate at 15–20 °C for 3–4 days to the late exponential phase. Occasionally slowly shake for 15-30 min (e.g., twice daily; max. at 30 rpm).
?. Place the cell cultures on a shaker (or a rotary platform) for 1–3 hours (15–20 °C) prior to isolation to detach all surface-adherent cells with gentle, continuous agitation or rocking (10–30 rpm, depending on the container).
?. Collect the cells at 2,000×g, 4 °C, for 5 min. Solubilize the pellets in 80 mL of OS at ambient temperature to wash away the remnants of serum, distribute into two 50-mL tubes, then spin (3,000×g, 4 °C, 5 min).
?. Solubilize each pellet in 30 mL of ice-cold SoHE and spin (8,000×g, 4 °C, 10 min).
?. Weigh the pellets (1-2 g/L of the culture are expected). Solubilize the pellets in ice-cold SoHE to 1 g (wet-weight) per 10 mL. From this step on, work on ice (unless otherwise stated).
?. While harvesting and washing the cells, prepare a two-step sucrose gradient. Into a 25×89 ultracentrifuge tube, add 6 mL of a HEPES-buffered 60% sucrose solution, then carefully overlay with 14 mL of HEPES-buffered 36% sucrose solution. Store in ice until use.For small-scale preparations, use 14×89 ultracentrifuge tubes and add 2 mL of 60% and 4.5 mL of 36% sucrose.Note that 36% sucrose corresponds to 1.2M, thus preserves the osmolarity of the SoHE buffer, which seems critical to avoid break-up of Diplonema's mitochondria.Adding EDTA to 2 mM into the HEPES-buffered sucrose solutions slightly decreases the contamination by cytosolic ribosomes.
?. Prepare 10–20 mL aliquots of cell suspension. Add protease inhibitor cocktail (20× in SoHE to 1× final) before transferring the cell suspension into a pre-cooled N2-cavitation chamber (on ice). Add a sterile stir bar. Apply 30 bar (435 psi/29.6 atm) N2 pressure for 3 min, while placing the chamber on a magnetic stirrer to ensure thorough mixing during the incubation.The volume of aliquots is imposed by the volume of the N2-cavitation chamber. Adjust according to the pressure chamber used.At 30 bar, which is the minimum pressure to open >80% cells, using SoHE at the concentration of 1.2 M sorbitol is necessary to avoid breaking up mitochondria and losing the contents of mitochondrial matrix into the solution. If one is only interested in mitochondrial membranes, it is possible to use 60 bar pressure in 0.65 M sorbitol to break virtually all cells.Prior to adding the cell suspension, verify the speed and the position of the chamber on the stirrer at which the bar rotates optimally.
?. Release the suspension from the chamber into a pre-cooled 50-mL tube. Put in ice and wait for the phase separation of the foam and suspension of broken cells. Repeat the steps #9 and #10 with the remaining aliquots.
?. Combine the suspensions from all aliquots (avoiding the foam) and overlay on the top of the sucrose gradients, distributing evenly the suspension. When using 25×89 ultracentrifuge tubes, spin using SW28 rotor (or equivalent) at 134,000×g (27,300 rpm), 4 °C, for 30–60 min.When working with 14×89 ultracentrifuge tubes use TH-641 rotor (or equivalent).For higher purity of mitochondria, two successive centrifugations (2,000×g, 4 °C, 10 min) can be performed to remove unlysed cells and nuclei prior to loading the mitochondria-containing supernatants on the sucrose gradient. (Note that the final yield will be >5 times lower.)
?. Collect the mitochondria-enriched band from the interface of 36%/60% sucrose using the syringe with an 18G needle.
?. To remove the sucrose (as well as cellular contaminants), dilute the mitochondrial fraction by adding with 3.5 volumes of SoH, gently, but thoroughly swirling the solution and then resuspending once or twice with a 18G-needle syringe stroke.To resuspend the collected, sucrose-containing mitochondrial fraction in SoH, one syringe stroke is usually enough. Being quick (and gentle) is essential, as an extended incubation with the buffer seems to favor the lysis of mitochondria and loss of the matrix into the supernatant after the subsequent centrifugation.If isolating just crude mitochondria and if sucrose content is irrelevant for downstream steps, flash-freeze the collected mitochondrial fraction without further manipulations.
?. Pellet the mitochondrial fraction (20,000×g, 4 °C, 10 min). Remove the supernatant and flash-freeze the final mitochondrial pellet in liquid nitrogen (or alternatively in dry ice).