Cell Biology Protocols

Reagents

Suspension buffer (SB): 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4

Gradient solutions: 35, 39 and 44% (w/v) sucrose in SB

Rat liver plasma membrane sheets (see

Protocol 4.26)

Include protease inhibitors in these solutions as required

Equipment

Dounce homogenizer (5–10 ml tight-fitting, Wheaton type A)

Phase contrast microscope

Syringe (10 ml) with metal filling cannula 1

Ultracentrifuge with 14–17 ml swingingbucket rotor and 13 ml fixed-angle rotor (with open-topped tubes)

3.Monitor the procedure using a phase contrast microscope (25× objective);

the large sheets of membrane should be converted principally to vesicles. 3

4.In tubes for the swinging-bucket rotor, use the syringe and metal cannula to

prepare discontinuous gradients from 3–5 ml each of the gradient solutions and layer 1–2 ml of the suspension on top. 4

5.Centrifuge at 196 000g for 3 h.

6.The canalicular membrane vesicles band at the sample/gradient interface; the basolateral (contiguous) domain at the 39/44% sucrose interface.

7.Collect each band with the syringe and cannula; dilute each with two volumes of SB and pellet at 200 000g for 30 min in the fixed-angle rotor.

8.Resuspend the pellet in SB for analysis.

Procedure

Carry out all operations at 0–4 ◦C.

1.Suspend the plasma membrane sheets in 2–5 ml of SB.

2.Disrupt the membranes using at least

30 vigorous strokes of the pestle of the Dounce homogenizer. 2

Notes

This procedure will take approx. 4 h.

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

2 A bath sonicator (80 W output) or a Polytron homogenizer (setting 8) is an alternative device for disrupting

the membrane sheets [2, 3]. Whatever method is used, the disruption must be regularly monitored. Start monitoring the preparation, for example, after 15 strokes of the pestle (Dounce homogenizer) or after five bursts of sonication.

3 The sheets of membrane, which are observed as characteristic dark Y- shaped structures (15–20 µm), should

be replaced by mass of black dots when disruption is complete.

4 The precise volumes will depend on the size of the centrifuge tube. For more information about the preparation of gradients see ref. 26.

Reagents

Anti-SC serum (or other primary antibody)

Medium A: 0.25 M sucrose, 50 mM TrisHCl, pH 7.4

Medium B: 2% (w/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, 30 mM Tris-HCl, pH, 7.4

Medium C: 1% (w/v) Triton X-100, 0.2% (w/v) SDS, 150 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl, pH 8.0

Medium D: as medium C minus the detergents

Non-immune serum

Protein–sepharose beads

Light mitochondrial supernatant: Protocol 4.8 as far as step 8 (use medium A for the homogenization medium) 1

Include protease inhibitors in solutions as required

Equipment

Dounce homogenizer (10 ml loose-fitting, Wheaton type B)

Shaking water bath at 24 ◦C

Microcentrifuge and microcentrifuge tubes

Ultracentrifuge with fixed-angle rotor for approx 13 ml tubes

Vortex mixer

Procedure

Carry out the isolation of the microsome fraction at 0–4 ◦C.

1.Centrifuge the supernatant (Protocol 4.8, Step 8) at 120 000g for 90 min to pellet the microsomes.

2.Resuspend the microsomes (using the Dounce homogenizer) in medium A (at 2 mg microsomal protein/ml).

3.Incubate 200 µl of a 60% (v/v) suspen-

sion of protein A–sepharose beads in medium B at 24 ◦C with 50 µl of antiSC serum (or non-immune serum) for 60 min.

4.Pellet the beads in a microcentrifuge and wash them four times in medium C, and a further two times in the medium D (use the vortex mixer to resuspend the beads in the media).

5.Incubate the beads in 500 µl of the microsome fraction for 2 h at room temperature.

6.Sediment the beads in a microcentrifuge and wash four times in medium A and then finally resuspend in this medium for further analysis.

Note

This procedure will take approx. 6 h (including preparation of the light mitochondrial supernatant).

1 In Protocol 4.8 do not omit the first centrifugation step at 800g. Failure to include this step will cause a significant loss of microsomal material due to entrapment by large, rapidly sedimenting particles and debris.

Reagents

Homogenization buffer (HM): 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4

Suspension buffer B (SB): as HM plus 5 mM EDTA

Hepes-buffered saline (HBS)

1 mM MgCl2

Gradient solutions: 34, 40, 54% (w/v) sucrose in 10 mM Tris-HCl, pH 7.4

Include protease inhibitors in solutions as required

Equipment

Low-speed refrigerated centrifuge with swinging-bucket rotor (10–15 ml tubes)

Nitrogen pressure vessel (Artisan Industries, Waltham, Mass, USA or Baskerville, Manchester, UK)

Syringe and metal filling cannula 1

Ultracentrifuge with (a) fixed-angle rotor

Procedure

Carry out all operations at 0–4 ◦C.

1.Rinse the cell monolayers once in HBS and once in HM.

2. Scrape the cells off the substratum with a rubber policeman into 5 ml of HM.

3.Homogenize the cells by nitrogen cavitation using nitrogen at 3795 kPa (550 psi) for 10 min and collect the

homogenate in a beaker (50 ml) covered with Parafilm , through which the delivery tube is sealed. 2 3

4.Allow the foam to subside by gentle stirring and then centrifuge the homogenate at 270g for 10 min.

5.Recentrifuge the supernatant at 920g for 10 min and carefully decant the supernatant.

6.Add 1 M MgCl2 to the supernatant to a final concentration of 10 mM.

7.Stir for 15 min before centrifuging at 2300g for 15 min.

8.Carefully decant the supernatant and centrifuge it at 170 000g for 45 min.

9.Resuspend the pellet in 0.5 ml of SB.

10.Using the syringe and cannula prepare a discontinuous gradient from 34, 40 and 54% (w/v) sucrose, 10 mM Tris-HCl, pH 7.4 (1.4, 1.7 and 1.4 ml

respectively) in tubes for the swingingbucket rotor. 4

11.Layer the suspension over the gradient and centrifuge at 68 000g for 4.5 h.

12.Remove the material at the interfaces with the syringe. The top band is enriched in Golgi membranes, the middle, in basolateral membranes and the bottom, in apical membranes.

13. Dilute the fractions with at least two volumes of HM and harvest the membranes by centrifugation at about 40 000g for 1 h.

Notes

This procedure will take approx. 9–10 h.

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

2 Assemble and operate the nitrogen cavitation vessel in accordance with the manufacturer’s recommendations.

Note that when the homogenate is collected, the valve on the delivery tube should be released only very slowly. For more information on the homogenization procedure see ref. 35.

3 It may be permissible to use other cell homogenization media, for example, the ball-bearing homogenizer or if neither of these machines is available, several passages through a gauge 23 or 25 syringe needle may suffice. The gradient fractionation is not known to be homogenization method sensitive. See ref. 35 for more information on the homogenization of cultured cells.

4 For more information on the preparation of gradients see ref. 26.

Reagents

OptiPrep

Homogenization medium (HM): 0.25 M sucrose,90 mMKOAc,2 mMMg(OAc)2, 20 mM Hepes-KOH, pH 8.0 1

Diluent:540 mMKOAc,12 mMMg(OAc)2,

120 mM Hepes-KOH, pH 8.0

Working solution (50% iodixanol): mix 5 vol. of OptiPrep and 1 vol. of diluent

Include protease inhibitors in HM and the diluent as required

Equipment

Ball-bearing homogenizer 2

Gradient collector 3

Low-speed refrigerated centrifuge with swinging-bucket rotor (10–15 ml tubes)

Phase contrast microscope

Refractometer (Abbe)´

Syringe and metal filling cannula 4

Ultracentrifuge with vertical rotor or nearvertical for 11–13 ml tubes 5

Procedure

Carry out all operations at 0–4 ◦C.

1.Homogenize the cells in HM in the ball-bearing homogenizer using four to

six passages. Monitor the efficacy of the homogenization by phase contrast microscopy.

2.Centrifuge the homogenate at 1000g for 5 min to pellet the nuclei. Decant and retain the post-nuclear supernatant (PNS).

3.Mix the working solution with the PNS to produce a final iodixanol concentra-

tion of 30% (format A) or 35% (format B). 6

4.Dilute the working solution with HM to produce solutions of 10 and 20%

iodixanol (Format A) or 10, 15, 20, 25 and 30 (Format B). 6

5.Layer equal volumes of the dense PNS and the chosen iodixanol solutions in

tubes for the vertical or near-vertical rotor. 7 8

6. Centrifuge at 353 000gav for 3 h using a slow acceleration program.

7.Allow the centrifuge to decelerate to rest from 2000 rpm without the brake or use a slow deceleration program.

8.Unload the gradient by tube puncture, upward displacement or aspiration from the meniscus in 0.5 ml fractions and assay for membrane markers [13, 14]. Measure the density profile

of a blank gradient using a refractometer. 9 10

Notes

1 Homogenization media often have to be tailored to a particular cell type; some alternatives are given in ref. 35.

2 Other homogenization methods may be permissible, such as Dounce homogenization or repeated passage through a gauge 23 or 25 syringe needle; for more information see ref. 35.

3 The type of unloading system that is permissible will depend on the tube type. Heat-sealed or crimp-sealed tubes must be unloaded by tube puncture, or possibly aspiration from the bottom of the gradient. Beckman Optiseal tubes, which are sealed with a central plastic plug, may also be unloaded by upward displacement with Maxidens or by aspiration from the meniscus using the Labconco Auto Densi-flow machine. See ref. 26 for more information.

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

5 The most convenient sealed tubes are the Optiseal for Beckman rotors. Some small volume fixed-angle rotors with a sedimentation path length

<25 mm may be suitable for making self-generated gradients.

6 Choose whichever format is most suitable; format A was used by Amieva et al. [14]; format B was used by Yeaman [13]. Both will lead to the formation of a gradient that is more or less linear after the centrifugation period.

7 A small volume of HM may be added to fill the tube to the required level.

8 Use the syringe and metal cannula to introduce the layers into the tube; see ref. 26 for more information.

9 Measurement of the density is not an absolute requirement but it makes comparison of banding patterns of basolateral and apical domain markers with published data easier. It also confirms the density profile, which should be linear over most of the gradient, becoming more steep in the densest region.

10 The peak density of the basolateral domain is approx 1.17 g/ml (about two-thirds of the way down the gradient), while that of the apical domain is approx. 1.08 g/ml (about a third of the way down the gradient).

PROTOCOL 5.5

Isolation of lipid rafts

Reagents

Homogenization medium (HM): 150 mM NaCl, 5 mM dithiothreitol (DTT), 5 mM EDTA, 25 mM Tris-HCl, pH 7.4 1

Lysis medium (LM): HM + 1% Triton X100 2

OptiPrep

Phosphate-buffered saline (PBS)

Triton X-100

Add protease inhibitors to HM as required

Equipment

Dounce homogenizer (tight-fitting, Wheaton type A) or syringe with 23 or 25 gauge needle 3

Gradient collector (optional) 4

Low-speed refrigerated centrifuge with swinging-bucket rotor (approx. 15 ml tubes)

Phase contrast microscope (for isolation from a post-nuclear supernatant only)

Syringe (5 ml) with metal cannula 5

Ultracentrifuge with swinging-bucket rotor (5 or 13 ml tubes) 6

Procedure

Carry out all operations at 0–4 ◦C.

Isolation from a total cell lysate

The procedure can be applied to any confluent cells from a single 3 cm culture

[15, 16]

dish. Scale up all volumes proportionally for larger amounts of cells.

1.Wash the cell monolayer twice with PBS and scrape into this medium using a rubber policeman.

2.Pellet the cells and resuspend in 0.2 ml of LM; then leave on ice for 30 min.

Isolation from a post-nuclear supernatant

The procedure is more convenient with larger numbers of cells (from one or two 9 cm dishes) because of the difficulty in homogenizing very small volumes.

1. Homogenize the cells in HM (up to

2 ml) using up to 20 strokes of the pestle of the Dounce homogenizer or up to the same number of passes through the syringe needle. 7

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

3.Decant the supernatant and adjust it to 1% Triton X-100 and leave on ice for 30 min.

Gradient separation

1. Add 2 vol. of OptiPrep to 1 vol. of either the homogenate or 1000g supernatant.

2.Dilute OptiPrep with LM to give 35, 30, 25 and 20% (w/v) iodixanol. 8

3.In tubes for the swinging-bucket rotor layer 0.6 ml each of the sample, the four

gradient solutions and LM to fill the tube. Modify or scale up the gradient for larger sample volumes 8

4.Centrifuge at 160 000gav for 4 h. 9

5.Collect the lipid rafts from the top interface or harvest the gradient in a

number of equal volume fractions and analyse as required. 10

Notes

The procedure will take approx. 5.5 h, using a 4 h centrifugation.

1 The isolation media used by both Oliferenko et al. [15] and Lafont et al. [16] were similar, although the level of DTT used by Oliferenko

et al. [15] was 1 mM rather than 5 mM and EDTA was omitted. Some proteins, which associate with lipid rafts, exhibit a Ca2+-dependence, so inclusion of a chelating agent may be detrimental to the study. DTT is often now omitted and sometimes the level of Triton X-100 in the discontinuous gradient is lower than in the sample layer. Protease inhibitors such as PMSF, leupeptin, antipain, aprotinin, etc. should be included in all of the media. Some of the variations in isolation media are given in Table 5.1 (column 2)

2 The level of Triton X100 is sometimes increased to 2% or reduced as

low as 0.1% (see Table 5.1). Occasionally Triton X-100 is substituted by CHAPS [48, 53]; sometimes the detergent is in the sample but omitted from the density gradient solutions.

3 Use whatever device is known to be suitable for the cell type; a ballbearing homogenizer might be a useful alternative. For more information on the homogenization of cultured cells see ref. 35.

4 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.

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

6 The smaller volume tubes are generally sufficient if a total cell lysate is to be used for the gradient input, but a larger volume tube may be easier for scale-ups or if a post-nuclear supernatant is to be prepared.

7 The details of the homogenization will have to be worked out for the specific cell type used. Use phase

contrast microscopy to monitor the homogenization.

8 Some of the variations in gradient format are given in Table 5.1 (column 3). Some workers have reduced the total number of layers to three (see Table 5.1); in this format the 5 ml tubes are capable of accommodating larger sample volumes. Lipid raft subdomains are best analysed using a different gradient format (see ref. 52 and Table 5.1). It may even be better to use a continuous gradient rather than a discontinuous one.

9 Oliferenko et al. [15] used a longer centrifugation time of 12 h at a slightly lower RCF (120 000gav). Because of the relatively short sediment path length of the rotor, 4 h at the higher RCF is probably satisfactory, but the centrifugation conditions may vary with the mode of preparation (see Table 5.1).

10 Depending on the resolution that is required it may be sufficient to use an automatic pipette to collect the gradient in four or five broad zones. Alternatively for higher resolution the gradient should be unloaded either by tube puncture, upward displacement or automatic aspiration from the meniscus (see note 4 and ref. 26). Always check on the distribution of raft and non-raft markers in the gradient to check that the centrifugation has achieved a satisfactory resolution and recovery of rafts.