Cell Biology Protocols

Reagents

OptiPrep

Diluent: 0.25 M sucrose, 6 mM EDTA, 120 mM Tricine-NaOH, pH 7.8

Working solution (50% iodixanol, ρ = 1.282 g/ml): mix 5 vol. of OptiPrep with 1 vol. of diluent

Suspension medium (SM): 0.25 M sucrose, 1 mM EDTA, 20 mM Tricine-NaOH, pH 7.6

Add protease inhibitors to diluent and SM as required

Equipment

Gradient maker (two-chamber) or Gradient Master

Gradient collector (optional)

Sonicator

Syringe and metal cannula (i.d. 0.8– 1.0 mm) 1

Ultracentrifuge with swinging bucket (14– 17 ml tube size)

Procedure

Carry out all operations at 0–4 ◦C.

1.Prepare a plasma membrane fraction by a suitable technique. 2

2.Suspend the plasma membrane in 2 ml

of SM in a suitable tube (approx.

1.5 cm diameter).

3.Place the tube in ice and introduce the tip of a sonicator probe (approx.

diameter 3 mm) to a point equidistant from the top and bottom of the suspension.

4.Sonicate twice for 6 s at a total power of 50 J/W per second, then allow to rest for 2 min before repeating the

sonication procedure twice more, i.e. a total of six sonication bursts. 3

5.Add 1.84 ml of 50% iodixanol work-

ing solution and 0.16 ml of SM. The final iodixanol concentration is 23% (w/v).

6.Produce two gradient solutions of 10% (w/v) iodixanol (ρ = 1.076 g/ml) and 20% (w/v) iodixanol (ρ = 1.125 g/ml)

by diluting the working solution with SM (1 + 4 and 2 + 3 v/v respectively). 4

7.Using a standard two-chamber gradient former or a Gradient Master produce a 9 ml linear 10–20% iodixanol

gradient in an approx. 13 ml tube for the swinging-bucket rotor. 4 5

8.Using the syringe and metal can-

ent; transfer to a new centrifuge tube and mix with 4 ml of working solution. 6 7

10.Produce two new gradient solutions of

5 and 15% (w/v iodixanol by diluting working solution with SM (1 + 9 and 3 + 7 v/v respectively)

11.Layer 1.0 ml of 15% and 0.5 ml of

5% iodixanol over the sample and centrifuge at 52 000g for 90 min. 8

12.Collect the caveolae-rich opaque layer that forms above the 15% iodixanol layer.

Notes

The procedure, excluding the preparation of the plasma membrane, will take approx. 4.5 h.

1 Metal ‘filling’ cannulas (i.d. 0.8– 1.0 mm) can be obtained from any surgical equipment supplies company.

2 The method used to prepare the plasma membrane will vary with the type of tissue or cell. As the source material may be a ‘routine’ cultured cell line such as human fibroblasts [54, 55], COS 7 [56, 57] or NIH 3T3 [18], a genetically modified line such as CHO-ldlA-7 [58, 59] or tissuederived cells such as lung microvascular endothelial cells [60, 61] or brain astrocytes [62], the operator should refer to the appropriate literature for a plasma membrane isolation technique. However, one of the methods described in Protocols 5.8 and 5.9 may be relevant.

3 If the sonicator does not conform to these parameters, it may be necessary to modulate the conditions in order to achieve the necessary fragmentation of the membrane. This can only be monitored by determining the caveolin and protein distribution in the first gradient (step 9).

4 The volume of the gradient needs to be calculated to fill the tube when

the 4 ml sample is underlayered. An approximately 9 ml gradient was used by Smart et al. [17]. The method can be scaled up or down as required, adjusting the volumes of sample and gradient proportionately.

5 If a gradient-forming device is not available, then prepare a discontinuous gradient from equal volumes of 10, 15 and 20% iodixanol and allow them to diffuse overnight at 4 ◦C. See ref. 26 for more information about making gradients.

6 If the volume of the gradient is increased, the volume occupied by the caveolae-containing fractions may also increase. As an approximation, the top third-to-half of the gradient should be removed. If the relative volumes of gradient and sample are significantly different it may be advisable to check the distribution of the caveolae by assaying fractions for caveolin by electroblotting (see Protocols 5.10–5.12 ).

7 Until the banding of the caveolin-rich, protein-poor material in the gradient is well established, it might be pertinent to collect the gradient by upward displacement with a dense medium; for more information on unloading gradients see ref. 26. Once the banding position of the caveolae has been established, the relevant part of the gradient can be removed using a syringe and cannula.

8 The volumes used in the second gradient may also have to be modified for larger volume tubes; it is probably good practice to increase the volumes of the sample and the two iodixanol layers, proportionally.

Reagents

Homogenization buffer (HB): 0.25 M sucrose, 10 mM Hepes-NaOH, pH 7.4

Gradient solutions: 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30 and 50% (w/v) sucrose in 10 mM Hepes-NaOH, pD 7.4 in D2O

Add protease inhibitors to all solutions as required

to 20 strokes of the pestle of the Dounce homogenizer, or up to 10 passes through the ball-bearing homogenizer or syringe. 3

2.Pellet the nuclei by centrifugation at 1000g for 10 min.

3.Using the syringe and cannula, prepare a discontinuous gradient from 1 ml of

each of the gradient solutions in tubes for the ultracentrifuge rotor. 4

Equipment

Dounce homogenizer (10 ml tight-fitting, Wheaton type A) or ball-bearing homogenizer or syringe fitted with 25 gauge needle

Gradient collector (upward displacement or collection from the meniscus) 1

Low-speed refrigerated centrifuge with swinging-bucket rotor for 10 ml plastic tubes

Phase contrast microscope

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

Procedure

Carry out all operations at 0–4 ◦C.

1.Suspend the cells in HB (no more than 5 ml) and homogenize using up

4.Layer 2 ml of the post-nuclear supernatant over the gradient and centrifuge at 160 000g for 3 h.

5.Unload the gradient by upward displacement (or aspiration from the

meniscus) into approx 0.3 ml fractions. 5

Notes

1 For gradient collection low-density end first use either the tube puncture device of the Beckman Fraction Recovery System to deliver a dense liquid such as Fluorinert (Sigma Aldrich) or Flutec-Blue (F2 chemicals Ltd, Preston UK) to the bottom of the tube or the Labconco Auto Densi-flow device to aspirate from the meniscus. For collection dense-end first use tube puncture. More information about gradient collection can be found in ref. 26.

2 Metal ‘filling’ cannulas (i.d. 0.8– 1.0 mm) can be obtained from any surgical equipment supplies company.

3 Monitor the progress of the homogenization by phase contrast microscopy; ideally no less than 90% cell lysis should be obtained, while the nuclei remain intact. More information about cell homogenization can be found in ref. 35.

4 Ref. 26 provides more information about the preparation of gradients.

5 Low density ER-Golgi intermediates band in the region 12.5–17.5% sucrose, while the ER itself bands around 22% sucrose.

Reagents

OptiPrep

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

Diluent: 0.25 M sucrose, 6 mM EDTA, 60 mM Hepes-NaOH, pH 7.4

50% (w/v) Iodixanol working solution (ρ = 1.272 g/ml): 5 vol. of OptiPrep + 1 vol. of diluent

Phosphate-buffered saline

Add protease inhibitors to HM and diluent as required

Equipment

Ball-bearing homogenizer, Dounce homogenizer (5 ml tight-fitting, Wheaton type A) or syringe with 25-gauge needle 2

Gradient maker (two-chamber or Gradient Master )

Gradient collector 3

Low-speed refrigerated and/or high-speed centrifuge with swinging bucket rotor for 15 ml tubes

Phase contrast microscope

Ultracentrifuge with swinging bucket rotor for approx. 13 ml tubes and fixed-angle rotor for 4–10 ml open-topped tubes (optional, see step 5 below) 4

Procedure

Carry out all operations at 0–4 ◦C.

1.Wash the cells twice in phosphatebuffered saline to remove the culture medium, and then once in the homog-

enization medium before resuspending in this medium. 5

2. Suspend the cells in a small volume of HM (0.5–5.0 ml) and disrupt them in a ball-bearing homogenizer, Dounce homogenizer or repeated passages through a fine syringe needle. 6

3.Centrifuge the homogenate at 1000– 3000g for 10 min; decant and retain the supernatant.

4.Resuspend the pellet in 0.5–2.0 ml of

HM; repeat step 3 and combine the two supernatants. 7

5.Optional step: Centrifuge the supernatant(s) at 100 000g for 40 min and

then resuspend the pellet in 1–2 ml of HM. 8

6.Prepare a 25% (w/v) iodixanol solution by mixing equal volumes of HM and the 50% iodixanol working solution.

7.Prepare 12 ml gradients in tubes for the swinging-bucket rotor from equal volumes of HM and the 25% iodixanol

solution using a two-chamber gradient maker or a Gradient Master. 9 10

8.Layer the vesicle suspension on top of

the gradient and centrifuge at 200 000g for 2–3 h. 10

9.Collect the gradient in 0.5 ml fractions by tube puncture or upward displacement and analyse by SDS-PAGE and electroblotting, probing with the appropriate antibodies to proteins in the membranes of interest (see Protocols 5.10–5.12 ). Prior to analysis, it may be necessary to concentrate the fraction either by precipitation with

trichloroacetic acid or by diluting it with 2 vol. of HM and pelleting by centrifugation at 100 000–150 000g for 40 min before resuspending in a small

the banding of the membrane compartments from a variety of cell types.

Notes

This procedure will take approx. 2 h.

1 With cultured cells the homogenization medium often has to be tailored to

the cell type. Sometimes an alternative buffer such as 10 mM triethanolamineacetic acid or the HM used in Protocol 5.4 achieves a better cell disruption. For more information see ref. 35.

2 The device of choice is the ballbearing homogenizer. Sometimes a syringe needle with a lower gauge number (wider orifice) will work satisfactorily. As with the homogenization medium, the homogenization protocol will need to be tailored to the cell type. See ref. 35 for more information.

3 For gradient collection low-density end first use either the tube puncture device of the Beckman Fraction Recovery System to deliver a dense liquid such as Fluorinert (Sigma Aldrich) or Flutec-Blue (F2 Chemicals Ltd, Preston UK) to the bottom of the tube or the Labconco Auto Densi-flow device to aspirate from the meniscus. For collection dense-end first use tube puncture. More information about gradient collection can be found in ref. 26.

4 Other swinging-bucket rotors or even vertical rotors may be used. Larger volume swinging-bucket rotors may require longer centrifugation times but vertical rotors will need shorter times. Most of the gradients have been run in 13 ml tubes but the gradients and sample volume can be scaled down as required.

5 Washing the cells with saline may be carried out at room temperature rather than at 4 ◦C if preferred.

6 Up to 10 cycles with the ball-bearing homogenizer, up to 15 strokes of

the pestle of a Dounce homogenization or up to 20 passages through a syringe needle should be sufficient. Always monitor the progress of homogenization by phase contrast microscopy.

7 Washing the low-speed pellet will release some trapped vesicles and improve yields.

8 The advantage of using a low-speed supernatant for the gradient input is that the procedure is quick and that vesicles in the supernatant are not further exposed to the shearing forces that are required to resuspend a 100 000g pellet. On the other hand, if it is important to remove any soluble cytosolic proteins then preparing a 100 000g pellet may be a useful step.

9 If neither of these devices is available then a continuous gradient can be prepared by diffusion of a discontinuous gradient [26].

10 The actual density range of the gradient may require modulation to suit the operator’s requirements. If the main interest, for example, is the Golgi and lower density fractions then a 0–20% or 0–15% iodixanol gradient may be more useful. Table 5.2 summarizes some of the variations for the 2–3 h centrifugations. Table 5.3 summarizes some of the 16–18 h centrifugation separations. A commonly used non-linear gradient is generated from layering the following iodixanol solutions: 1 ml of 2.5%, 2 ml each of 5, 7.5 and 10%, 0.5 ml of 12.5%, 2 ml of 15%, and 0.5 ml each of 17.5, 20 and 30%. These gradients appear particularly good for resolving the TGN [76–80].

Reagents

OptiPrep

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

Diluent I: 0.25 M sucrose, 6 mM EDTA, 60 mM Hepes-NaOH, pH 7.4 1

Homogenization medium II (HMII): 130 mM KCl, 25 mM NaCl, 1 mM EGTA, 25 mM Tris-Cl, pH 7.4 2

Diluent II: 130 mM KCl, 25 mM NaCl, 6 mM EGTA, 150 mM Tris-Cl, pH 7.4 2

Working solution of 50% (w/v) iodixanol: 5 vol. of HM +1 vol. of diluent I or II 3

Equipment

Ball-bearing homogenizer, Dounce homogenizer (5 ml tight-fitting, Wheaton type A) or syringe with 25-gauge needle 4

Gradient collector 5

Gradient maker (two-chamber or Gradient Master ), for continuous gradient only 6

Low-speed refrigerated and/or high-speed centrifuge with swinging bucket rotor for 15 ml tubes

Syringe (5 ml) and metal cannula, for discontinuous gradient only 7

Ultracentrifuge with swinging-bucket rotor for approx. 13 ml tubes 8

Procedure

Carry out all operations at 0–4 ◦C.

1.Produce a cell homogenate from the chosen tissue or cells using HM (I or

II)using steps 1–2 of Protocol 5.8 .

2.Centrifuge the homogenate at 1000g for 10 min.

3.Centrifuge the supernatant from step 2 at 3000g for 10 min; decant and retain the supernatant.

4.For a discontinuous gradient: Prepare

10 ml each of 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 and 30% (w/v) iodixanol solution by mixing HM with the 50% iodixanol working solution containing the appropriate diluent (I or II).

5.For a continuous gradient: Prepare 20 ml of 10 and 30% iodixanol solutions as in step 4.

6.In 13 ml tubes for the swinging-bucket rotor (discontinuous gradient): layer 1.2 ml each of the nine gradient solutions using the syringe and metal

cannula; (continuous gradient): use a two-chamber gradient maker or Gradient Master to make an approx. 12 ml gradient from equal volumes of the 10 and 30% iodixanol solutions. 9

7.Layer the supernatant from step 3 on top of the gradient.

9.Collect the gradient in 0.5 ml fractions by tube puncture or upward displacement and analyse by SDS-PAGE and electroblotting, probing with the appropriate antibodies to proteins in the membranes of interest (see Protocols 5.10–5.12 ). Prior to analysis, it may be necessary to concentrate the fraction either by precipitation with trichloroacetic acid or by diluting it with 2 vol. of HM and pelleting by centrifugation at 100 000–150 000g for

40 min before resuspending in a small volume of HM. See the references in Table 5.4 for details of the banding of the membrane compartments from a variety of cell types.

Notes

This procedure will take approx. 4 h.

1 This is general purpose HM and diluent and has been used for many types of cultured cells and tissues.

2 This salt-containing HM and diluent was used for neuroblastoma cells [27] and brain tissue [81] but may be particularly applicable to any cells and tissues that release proteins that may cause agglutination of membranes. See ref. 35 for other homogenization media.

3 Use the appropriate diluent for the chosen HM.

4 The device of choice is the ballbearing homogenizer. Sometimes a syringe needle with a lower gauge number (wider orifice) will work satisfactorily. As with the homogenization medium, the homogenization protocol will need to be tailored to the cell type. See ref. 35 for more information.

5 For gradient collection low-density end first use either the tube puncture device of the Beckman Fraction Recovery System to deliver a dense liquid such as Fluorinert (Sigma Aldrich) or Flutec-Blue (F2 Chemicals Ltd, Preston UK) to the bottom of the tube or the Labconco Auto Densiflow device to aspirate from the meniscus. For collection dense-end first use tube puncture. More information about gradient collection can be found in ref. 26.

6 If neither of these devices is available then a continuous gradient can be prepared by diffusion of a discontinuous gradient [26].

7 Metal ‘filling’ cannulas (i.d. 0.8– 1.0 mm) can be obtained from any surgical equipment supplies company.

8 Other swinging-bucket rotors or even vertical rotors may be used. Larger volume swinging-bucket rotors may require longer centrifugation times but vertical rotors will need shorter times. Most of the gradients have been run in 13 ml tubes but the gradients and sample volume can be scaled down as required.

9 See ref. 26 for information on making discontinuous gradients. Continuous gradients can be prepared by allowing a discontinuous gradient of equal volumes of 10, 20 and 30% iodixanol to diffuse overnight.

10 These are the most widely reported centrifugation conditions; see Table 5.4 for examples of separations using the various conditions described in this protocol.