Cell Biology Protocols

11.Also carefully aspirate the loosely packed pinkish material on top of the more compact brown pellet and wipe the inside of the tube with a tissue to remove any adhering lipid.

12.Add 10 ml of buffer to each tube and crudely resuspend the pellet using a glass rod.

13.Complete the resuspension using two to three gentle strokes of the pestle of the Dounce homogenizer.

14.Make up to the original volume with buffer and decant into new tubes.

15.Repeat steps 9–14 three times. 10

16.Finally, resuspend the purified heavy mitochondria in a suitable medium compatible with the subsequent analysis.

Notes

This procedure will take approx. 1.5 h.

1 For other tissues, such as beef heart, a sucrose/succinate/EDTA/Tris buffer is common – see ref. 72.

2 Steps 5 and 7 could be executed using a high-speed centrifuge rather than a low-speed centrifuge.

3 Starving the animal must be carried out in accordance with national animal care requirements.

4 For respiratory studies, it is obviously important to avoid any anaesthetic techniques in sacrificing the animal. Decapitation is a useful alternative, which also serves to exsanguinate the liver, but whatever technique is used, it must be carried out by a trained, licensed individual.

5 Do not force the pestle through any compacted tissue pieces at the bottom of the homogenizer; lower the glass vessel to resuspend the tissue in the vortex from the revolving pestle.

6 It is advisable not to fill the tubes of any rotor, a swinging bucket rotor in particular, completely full, because of the problems associated with use of these rotors (see ‘Differential centrifugation’ above).

7 Washing the nuclear pellet will recover any trapped mitochondria.

8 Removal of this supernatant with a syringe, rather than by decantation, reduces contamination by nuclei from the pellet.

9 The presence of free fatty acids can cause uncoupling, so it is important to remove as much as possible of the lipid.

10 The washing procedure removes contaminating lysosomes.

Reagents

Homogenization medium (HM): 0.25 M sucrose, 1 mM EDTA, 10 mM HepesNaOH, pH 7.4 1

Add protease inhibitors to the above medium as required

Phosphate buffered saline (cells only)

Equipment

Low-speed refrigerated centrifuge with swinging-bucket rotor for 30–40 ml tubes 2

High-speed centrifuge with fixed-angle rotor for 30–40 ml tubes

For liver: Potter-Elvehjem (Teflon and glass) homogenizer (30–40 ml) attached to a high-torque overhead motor (thyristor controlled) 3

For cells: Ball-bearing homogenizer or syringe with 23or 25-gauge needle 3

Dounce homogenizer (loose fitting, Wheaton type B)

Syringe (20 ml) with metal cannula (internal diameter approx. 0.8 mm) 4

chopper) and transfer to the PotterElvehjem homogenizer with HM (use 10 ml medium for every 2.5 g tissue). Homogenize using approx. six strokes of the pestle (500–700 rpm). 5

2. For cells: Wash 1–3 × 108 cells in 5 ml of phosphate buffered saline and again with 5 ml of HM. Suspend the cells in 3 ml of HM and homogenize in a ball-bearing homogenizer using five passes. 6 7

3.Pellet the nuclei, cell debris and any unbroken cells by centrifuging at 800g for 10 min.

4.Decant or aspirate (use a syringe and cannula) the supernatant and retain on ice.

5.Resuspend the pellet in 10 ml (5 ml for cells) of HM using two to three

gentle strokes of the pestle of a loosefitting Dounce homogenizer. 8

6.Repeat the centrifugation and combine the supernatants.

7.Centrifuge the combined supernatants at 3000g for 10 min. 9

8.Centrifuge the supernatant from step 7 at 15 000g for 10 min. 10

Procedure

Carry out all operations at 0–4 ◦C.

1.For liver: Mince the tissue very finely with scissors (or with a tissue

9.Resuspend the light mitochondrial pellet in 10 ml (5 ml for cells) of HM using two to three gentle strokes of

the pestle of a loose-fitting Dounce homogenizer. 8

10.Centrifuge at 15 000 g for 10 min.

11.Resuspend the pellet in a suitable medium for analysis or further processing. Use this for Protocols 4.10, 4.12, 4.15 and 4.17.

Notes

This procedure will take approx. 2 h.

1 Any suitable buffered isoosmotic 0.25 M sucrose solution may be used, with or without EDTA. If the aim is to isolate peroxisomes, include 0.1% ethanol.

2 The pelleting of the nuclei, etc. (step 3), although often carried out in a lowspeed centrifuge, may alternatively be performed in the high-speed fixedangle rotor.

3 For more information on homogenizing cells and tissues, see ref. 2.

4 Metal ‘filling’ cannulas can be obtained from any surgical equipment supplies company.

5 Most soft tissues can be homogenized in a Potter-Elvehjem homoge-

nizer, but alternatives such as the Polytron homogenizer have been used.

6 For the isolation of organelles from cultured cells, the ball-bearing homogenizer is considered to offer the gentlest means of disruption [14]. Alternatives are passage through a syringe needle or a tight-fitting Dounce homogenizer.

7 Always monitor the efficacy of the homogenization by phase contrast microscopy.

8 Resuspension of the pellet must be carried out under the mildest of conditions.

9 This step to remove the heavy mitochondria may be omitted.

10 A variety of centrifugation conditions have been used for isolation of the light mitochondrial fraction; RCFs of 12 000–20 000g and times of 10–20 min are the most common. Check the recovery of the required organelle and modify as required. Always use the minimum g-force to give an adequate recovery.

Reagents

Yeast spheroplast preparation in sorbitol buffer 1

Nycoprep Universal (60% w/v Nycodenz )

Nycodenz working solution: 50% w/v Nycodenz

Sorbitol buffer: 0.6 M sorbitol, 20 mM Mes-KOH, pH 6.0

Sorbitol buffer (2×): 1.2 M sorbitol, 40 mM Mes-KOH, pH 6.0

Suspension buffer (SB): 0.6 M sorbitol, 20 mM Hepes-KOH, pH 7.4

Add protease inhibitors as required to the buffers

Equipment

High-speed centrifuge with 8 × 50 ml fixed-angle rotor

Ultracentrifuge with swinging-bucket rotor (approx. 12–13 ml tubes)

Dounce homogenizer (approx. 40 ml tightfitting, Wheaton type A)

Dounce homogenizer (approx. 40 ml loose-fitting, Wheaton type B)

Syringe (20 ml) with metal cannula (internal diameter approx. 0.8 mm) 2

Procedure

Carry out all operations at 0–4 ◦C.

1.Homogenize the spheroplasts using 15 strokes of the pestle of the tight-fitting Dounce homogenizer.

2.Dilute with an equal volume of sorbitol buffer and centrifuge in the fixedangle rotor at 1500g for 5 min.

3.Decant the supernatants and retain.

4.Resuspend the pellets to the original volume with sorbitol buffer and repeat steps 1 and 2.

5.Combine all the supernatants and centrifuge at 12 000g for 10 min.

6.Resuspend the crude mitochondrial pellets in approx 40 ml sorbitol buffer using a few gentle strokes of the pestle of the loose-fitting Dounce homogenizer.

7.Remove any residual nuclei and debris by centrifugation at 1500g for 5 min.

8.Remove the supernatants using the

syringe and cannula, taking care not to disturb the loosely packed pellet. 3

9.Centrifuge the supernatants at 12 000g for 10 min and resuspend the pellets

(loose-fitting Dounce homogenizer) in approx 4 ml of sorbitol buffer. 3

10.Prepare two Nycodenz solutions of

18 and 14.5% (w/v) from 25 ml of sorbitol buffer (2×) and 18 ml (and 14.5 ml) of the 50% Nycodenz

working solution; make up each to 50 ml with water.

11.In tubes for the swinging-bucket rotor load 5 ml of each of the Nycodenz solutions and approx. 2 ml of the crude mitochondrial suspension (top

up with sorbitol buffer if necessary).

4

band at the interface of the two Nycodenz layers.

13.Harvest the band with the syringe and metal cannula; dilute with 4 vol. of SB; centrifuge at 12 000g for 15 min and resuspend the pellet in SB.

Notes

This procedure will take approx. 2 h.

1 The spheroplast suspension from approx 25 g of packed yeast cells (wet weight) should be approx. 35 ml.

2 Metal ‘filling’ cannulas can be obtained from any surgical equipment supplies company.

3 Decantation of the supernatant may lead to contamination from the pellet material.

4 See ref. 18 regarding the preparation of discontinuous gradients.

Reagents

Homogenization medium (HM): 0.25 M sucrose, 1 mM EDTA, 10 mM HepesNaOH, pH 7.4 1

Nycoprep Universal

Nycoprep Universal diluent (NUD): 6 mM EDTA, 60 mM Hepes-NaOH, pH 7.4

Nycodenz working solution (50%, w/v): Mix 5 vol. of Nycoprep Universal with 1 vol. of NUD.

Light mitochondrial fraction (see Protocol 4.8 )

Include protease inhibitors in these solutions as required

2.Resuspend the washed light mitochondrial pellet (LMP) in HM (4–10 ml)

and mix with an equal volume of Nycodenz working solution.

3.For 36–38 ml tubes, use a syringe with

metal cannula to underlayer (low density end first) the following Nycodenz solutions: 2 ml of 20%, 7 ml of 23%, 10 ml

of LMP in 25%, 7 ml of 30%, and 5 ml each of 34 and 40%. 4 5 6

4. After centrifugation at 95 000 g for

2 h harvest the mitochondria from the 25/30% Nycodenz interface or unload the entire gradient in a series of equal volume fractions prior to analysis. 7

Ultracentrifuge and swinging-bucket rotor with a tube capacity of approx. 38 ml, smaller volume 17 ml or 13 ml tubes are also acceptable

Syringe (10 ml) with metal cannula (internal diameter approx. 0.8 mm) 2

Procedure

1.Dilute the Nycodenz working solution

with HM to produce gradient solutions with Nycodenz concentrations of 40,

34, 30, 23 and 20% (w/v). Keep these solutions at 0–4 ◦C. 3

The procedure will take approx. 4.5 h, including preparation of the LMP.

1 Use the same HM as used in Protocol 4.8.

2 Metal ‘filling’ cannulas can be obtained from any surgical equipment supplies company.

3 The low-density end of the gradient is probably ineffective at resolving any Golgi membranes from the lysosomes. For this an additional layer of 15% Nycodenz might be layered on top.

4 For methods on the best ways of constructing discontinuous gradients, see ref. 18.

5 Top up the tubes, if necessary, with either 20% Nycodenz or HM, according to the manufacturer’s recommendations for tube filling. If using the additional layer of 15%

Nycodenz , use 2 ml of this and reduce the layer of 40% Nycodenz to 3 ml.

6 Scale down the volumes proportionally for smaller tubes.

7 See ref. 18 for more information on harvesting and analysing gradients.

Reagents

Ethyl acetate/ethanol/trichloroacetic acid (EET) 5 : 5 : 1 (v/v/w)

p-Iodonitrotetrazolium violet (2.5 mg/ml in phosphate buffer)

Phosphate buffer pH 7.4 (50 mM)

Substrate: 0.01 M succinate in phosphate buffer

Equipment

Microcentrifuge with 1.5–2.0 ml tubes

Spectrophotometer (visible wavelength) with 1 ml cuvettes (glass or EETresistant plastic)

Procedure [73]

1.Dilute fractions from a gradient with an equal volume phosphate buffer and

sediment in a microcentrifuge at 4 ◦C for 5–10 min.

2.Resuspend the pellets by vortex mixing in 0.3 ml of substrate. 1

3. After incubation at 37 ◦C for 10 min add 0.1 ml of INT and incubate for a further 10 min. 2

4.Stop the reaction by addition of 1 ml of EET.

5.Centrifuge any precipitate in a microcentrifuge for 2 min and measure the

absorbance of the supernatant at 490 nm against a suitable control. 3 4

Notes

The procedure will take approx. 1 h.

1 Rather than centrifuging and resuspending a gradient fraction in substrate solution, small volumes of gradient fraction (<50 µl) may be added directly to the substrate solution. Nycodenz does not interfere with the enzyme assay.

2 This incubation time may be extended if the concentration of enzyme is low.

3 Ideally, for each test incubation, a control incubation should be set up in which the substrate solution is replaced by phosphate buffer. In most cases, however, a single control containing substrate solution, buffer, INT and EET is sufficient.

4 The molar extinction coefficient of reduced INT is 19 300.

Reagents

Homogenization medium (HM): 0.25 M sucrose, 1 mM EDTA, 10 mM HepesNaOH, pH 7.4 1

Nycoprep Universal

Nycoprep Universal diluent (NUD): 6 mM EDTA, 60 mM Hepes-NaOH, pH 7.4

Nycodenz working solution (50%, w/v): Mix 5 vol. of Nycoprep Universal with 1 vol. of NUD

Add protease inhibitors to solutions as required

Light mitochondrial fraction (see Protocol 4.8 )

Equipment

Ultracentrifuge with swinging-bucket rotor with a tube capacity of approx. 38 ml (smaller volume 17 ml or 13 ml tubes are also acceptable).

Syringe (10 ml) with metal cannula (internal diameter approx. 0.8 mm) 2

Procedure [45]

Carry out all operations at 0–4 ◦C.

1.Dilute the Nycodenz working solution

with HM to produce gradient solutions with Nycodenz concentrations of 30,

26, 24 and 19% (w/v). Keep these solutions at 0–4 ◦C.

2.Resuspend the washed light mitochon-

drial pellet in HM (4–10 ml) and mix with 2 vol. of Nycodenz working solution.

3.For 36–38 ml tubes: Transfer 8 ml of 19% Nycodenz into each tube and underlayer, using a syringe and metal

cannula, 7 ml each of the 24, 26 and 30% Nycodenz and 8–9 ml of the

light mitochondrial pellet suspension.

3 4 5

4.Centrifuge at 95 000g for 2 h and then

harvest the lysosomes from the 19/24% Nycodenz interface or unload the

entire gradient in a series of fractions prior to marker analysis. 6

Notes

The procedure will take approx. 4.5 h, including preparation of the LMP.

1 Use the same HM as used in Protocol 4.8.

2 Metal ‘filling’ cannulas can be obtained from any surgical equipment supplies company.

3 For methods on the best ways of constructing discontinuous gradients, see ref. 18.