Separation and application of lipid rafts

Summary

Lipid rafts are important in immunology because T-cell receptors and several other important T-cell signaling proteins are present in lipid rafts or are recruited to lipid rafts during signaling. Similarly, the high-affinity IgE receptor on basophils is also recruited into lipid rafts during signaling.

Author: J.E. Colligan et al, Translated by Xuitao Cao et al. This experiment is from the "Comprehensive Immunology Laboratory Guide".

Operation method

Separation and application of lipid rafts

Move

The basic scheme is to prepare detergent-resistant membranes by sucrose gradient flotation and to analyze the proteins by immuno-imprinting. Materials

Cells, walled or non walled

N E /P buffer, alone or containing one or more of the following components:

1 %, 2 % or 5 % (W V ) TritonX-IOO

0.lmol/L sodium carbonate, pHll

5 %, 35 % or 80 % (m/V) sucrose

60 m m o l /L octyl glucoside

T N E buffer

Decontaminant-compatible protein assay kits (e.g., Bio-Rad)

2 X or 6 X S D S / Sample Buffer

Acetone (optional), ice cold

Ether (optional)

IO c m tissue culture dish (for adherent cells)

22 G needle

I m l and 5 m l syringes

S W 4 1 rotor and ultracentrifuge tube (B e c k m a n )

For adherent cells, cell lines that do not need to be compared

la. Spread cells in 2 I O c m tissue culture dishes and grow until cells are confluent. For D R M lipid analysis (see Supporting Scheme 3), inoculate cells to 5 culture dishes or 10 dishes to quantify phosphatidylserine, phosphatidylinositol, or sparsely available gangliosides.

2a. Cells were lysed with I m l of T NE/P buffer containing 1 or 2 % Triton X-100 on ice for 20 min. 0.lmol/L sodium carbonate, p H l l, was added to the lysis buffer, if necessary, to better quantify the D RM lipid yield, to increase the D RM lipid yield, or to reduce the binding of peripheral or cytoskeletal proteins to D RM. Carbonate should not be used when analyzing enzyme activity in D R M .

3a. Use a cell scraper or rubber scraper to scrape off Petri dish cell lysates and collect them in a tube.

4a. If the lysis buffer contains carbonate, pass the lysate through a 5 m l syringe with a 22 G needle 1 0 times (keeping the lysate product cold) for shearing the D N A to ensure that the D R M is moved to the correct gradient.

For adherent cells, cell lines to be compared between cells

lb. To compare the abundance and yield of D R M between two different cell types, culture the cells of the different cell types in Petri dishes as shown in Step Ia, inoculate a separate dish with each cell (to determine the protein content of the culture), and ensure that the number of cells is the same in each dish.

2b. Rinse one dish of each cell type twice with 5 ml of T NE buffer and lysed the cells with I ml of T NE/P buffer containing 1 % TritonX-100 or 0. lmol/L sodium carbonate. The viscous cell lysate is immediately transferred to a 1.5 m l microcentrifuge tube and passed through a 22 G needle-attached Imi injector 10 times (or until the viscosity disappears) to shear the D N A. The lysate is then centrifuged at maximum speed. The lysate is then centrifuged at maximum speed.

3b. Determine the protein content of a 5- to IOul aliquot of lysate using a detergent-compatible protein assay kit. T NE buffer was mixed with T NE buffer containing 2 % or 5 % TritonX-100 to prepare T NE/P buffer containing sufficient TritonX-100 to give a 10:I (m/m) detergent:protein mix. To increase the yield of proteins that are weakly bound to lipid rafts, a detergent:protein mix of 2:5 (m/m) is used.

4b. Add I m l of ice-cold T N E /P buffer containing 1 % Triton X -IOO to the remaining petri dishes and lysed the cells on ice for 20 min. 0.lmol/L sodium carbonate, p H l l , was added to the lysis buffer for better quantification of the D R M lipid yields, to increase D R M lipid yields, or to reduce the binding of peripheral or cytoskeletal proteins to D R M . Carbonate cannot be used when analyzing enzyme activity in D R M . Follow Step 3a; if necessary, follow Step 4a.

For non-adherent cells

Ic. Culture approximately 5X 107 cells and centrifuge at 4°C, 200 g for 5 min. For cell-to-cell comparisons, equal numbers of cells (of the same origin) should be used. For lipid analysis (see Additional protocol 3), use 2X 108 non-adherent cells.

2c. Resuspend the cells in 5 ml of ice-bath TNA buffer and rewash twice.

3c. Lysed the cells with 2 ml of TNE/P buffer containing 1 % Triton X-100 on ice for 20 min. 0.lmol/L sodium carbonate, pH l, was added to the lysis buffer to better quantify the lipid yield of D RM, to improve the lipid yield of D RM or to reduce the binding of peripheral or cytoskeletal proteins to D RM. In analyzing the enzymes in D R M
activity in D R M, carbonate should not be used.

4c. If necessary, follow step 4a.

5 . Mix the lysate with ice-cold TNE/P buffer containing 80 % sucrose. For lysates from adherent cells, add 1.25 times the total volume of lysis buffer to the dish as described above (i.e., 1.25 ml for lysates collected in Steps 3a or 4b). For lysates from non-adherent cells, add 2.5 ml. Mix thoroughly and allow to stand for a few minutes to minimize foaming.

6 . Transfer to one or more S W 4 1 ultracentrifuge tubes, removing any foam that may be present. Do not exceed 5 ml per tube. carefully cover the lysate/sucrose complex with 6 to 7 m l of ice-cold T N E /P Buffer Liquid containing 3 5 % sucrose (the layering should be as wide as possible). start pouring the liquid with a watchful eye to make sure that it can float to the top. If it does not float, remove the mixture from the microcentrifuge tube and mix thoroughly. If necessary, add a small amount of 8 0 % sucrose.

7 . Fill the centrifuge tube to approx. 3 m m from the top with chilled TNE/P buffer containing 5 % sucrose. Centrifuge for > 3 h (to 24 h) at 4°C and 10000 Qg.

Detection of protein gradient distribution

8a. Collect the components on the gradient in sections from the bottom, lml per collection. add 2 X or 6 X SDS/Sample Buffer and analyze 50-100ul of the components by SDS-PAGE (Unit 12.3) and immuno-imprinting (Unit 12.5).

For immunoblotting without immunoprecipitation

8b. Collect a visible band from the tip of the centrifuge tube at the 5%/35% sucrose junction with a 5 ml syringe 22 G needle in 1 S W 4 1 ultracentrifuge tube. Dilute the membrane composition with ice-cold T NE/P buffer until the tube is filled. Centrifuge 111 at 4°(:, 100 00(^.

9b. Discard the supernatant. Pipette the sediment with 100~2 0 0 4 T N E /P buffer and transfer to a microcentrifuge tube. Wash the ultracentrifuge tube 3 times with 200 ul of TNE/P buffer and scrape the bottom of the tube to collect sediment fragments. Combine these washes with the sediment.

10b. Centrifuge at 4°C for IOmin at maximum speed to precipitate D R M . Discard the supernatant. D R M is dissolved directly in 2X or 6X SDS/Sample Buffer and analyzed by SDS-PAGE (Unit 12.3) and immunoblotting (Unit 12.5).

For low molecular mass proteins

8c. Prepare D RM precipitate according to steps 8b~IOb without solubilizing with SDS/sample buffer. Add I ml of ice-cold acetone to the D RM precipitate, vortex to mix and incubate on ice for lOmin. Centrifuge at 4°C for lOmin at maximum speed and discard the acetone.

9c. Add I ml of diethyl ether to the tube at room temperature, vortex well, and precipitate as described above. Remove the ether and air-dry. D R M protein is solubilized with 2 X or 6 X SDS/Sample Buffer and analyzed by SDS-PAGE (Unit 12.3) and immunoblotting (Unit 12.5).

Immunoblotting after immunoprecipitation

8d. Prepare D RM precipitate according to steps 8b to Iob without dissolving in SDS/sample buffer. D R M was dissolved with I ml of TNE/P buffer containing 1 % Triton X-IOO and incubated at 37°C for 5 min, or with Iml of TNE/P buffer containing 60 m m o l/L octylglucoside and incubated on ice for 20 min.

9d. Centrifuge at 4°C for 5 min at maximum speed to precipitate any remaining insoluble material. The clarified soluble D R M is transferred to a new tube and the target protein is recovered by the immuno-coimmunoprecipitation assay (Unit 12. 2) and analyzed by S D S - P A G E (Unit 12. 3) and immunoimmunoimprinting (Unit 12. 5).

Option to prepare detergent-resistant membranes by centrifugation Additional materials

Dissolution in S D S
3a Resuspend the lysed precipitate of Triton X -IOO with 100ul of precipitate lysate. Pass the mixture through an Iml injector with a 22 G needle (to shear the DNA) until homogeneous. Dilute with I ml of ice-cold TNE/P buffer containing 1 % Triton X-100, centrifuge at maximum speed for 2 min at 4°C, retain the supernatant and discard the remaining insoluble material.

4a. For comparison of the two fractions, the initial Triton X-100 supernatant (step 2) is adjusted to a final concentration of 0.1% SDS using a 20% SDS reservoir. The final concentration of 0.1% SDS was adjusted to a final concentration of 0.1% SDS with 20% SDS stock solution.
Equal amounts of the two fractions are analyzed by immunoprecipitation (Unit 12.2), SDS-PAGE (Unit 12.3) and immunoblotting (Unit 12.5).

Solubilization in octylglucoside

3b. Lysate the precipitate with I ml of ice-cold TNE/P buffer containing 60 mmol/L octylglucoside by resuspending Triton X-100 on ice for 20 min in order to solubilize DRM protein. Vortex to mix and centrifuge at 4°C for 2 min at maximum speed, retain the supernatant and discard the precipitate.

4b Analyze equal amounts of this fraction and the original Triton X-100 lysate supernatant (Step 2) by immunoprecipitation (Unit 12.2), SDS-PAGE (Unit 12.3), and immunoblotting (Unit 12.5).

Supplementary Option 1: Analysis of detergent-resistant membrane proteins by pulse-tracking methods Additional material
Supplementary Scheme 2 Detection of all components of membrane proteins resistant to detergents by radiolabeling Additional material

PBS, sterile

Methionine-free, dialyzed serum-containing medium (see Appendix 1 for method of dialyzing serum; dosage depends on cell type).

[ ^35S ] Methionine

1 . Inoculate wall-adherent cells in I o c m tissue culture dishes. If necessary, inoculate the cells to be tested in two dishes, but label only one of the dishes (for non-adherent cells, inoculate 5 X ¹⁰⁶ cells, separate from the other).

Supplementary Option 3 Quantitative High Performance Thin Layer Chromatography (HP-TLC) for DRM Lipid Analysis Materials

Methanol

chloroform

I : 1 and 2 : I (V/V) Chloroform/Methanol

Solvent A : 30 : 60 : 8 (V/V A O Chloroform/Methanol/Water)

Silylation reagent (Sigmacote, Sigma)

DEAE dextran gel (Sephadex) suspension (see Supporting Program 4)

Solvent B : 30 : 60 : 8 (V 7V7 V ) chloroform/methanol/0-8mol/L sodium acetate

8 : 4 : 3 Chloroform/methanol/0. lmol/L sodium chloride

0.lmol/L sodium chloride

Solvent C : 3 : 48 : 47 (W V /V) Chloroform/Methanol/0.lmol/L NaCl

Solvent D : 3 : 48 : 47 (V/V/V) Chloroform/Methanol/Water

Nitrogen

Reversed phase C 18 column (see Auxiliary Program 5)

Neutral Lipid Standard

Acid Lipid Standard

Acetic acid

Formic acid

Hexane

isopropyl ether

Lipid developing reagent

S W 2 8 Rotor and Ultracentrifuge Tubes (Beckman)

15 ml glass tubes with Teflon lined caps

High power (Laboratory Supplies, Model G S P 1T) or standard bath ultrasonic breakers

24 m m Whatman G F /C glass fiber filter

Filtration device: glass microanalytical filter (Fisher), or 24m m Buchner funnel with rubber gasket inserted into 125m l side arm flask

125 ml flat-bottomed flasks and tubes with ground glass necks

Rotary evaporator

Glass wool

One-time IO ml wide-mouth glass pipettes

Glass rods

IO m l silanized pipettes

5 m l conical glass tube with T e f lo n lined cap

125 ml side arm flask with single hole stopper

High Performance Thin Layer Chromatography (HP-TLC) plate (Merck) 10 c m X 20 c m without fluorescent indicator

Thin-layer chromatography cylinder with glass lid

Preheated oven to 100°C, 110°C, 180°C

Improved IOjL d Hamilton syringe: syringe with beveled edges reduces the loading angle by half, facilitating the loading of small amounts of drops

IOOm l graduated cylinder with ground glass stopper

Absorbent paper cut to the size of a chromatography cylinder

Hair dryer with cooling device

0.5~I c m thick wrong block

Optical Densitometer

Note : All glassware in contact with D E A E Glucose Gel should be silanized before use. After the glass wool is loaded , silanize the glass wool together with the column.

1 . Prepare 10 bottles of lysate from walled cells in IO c m dishes (see Basic Plan, Steps Ia to 4a) or lysate from 2 X ^IO8 non-walled cells (see Basic Plan, Steps Ic to 4c).

2 . Prepare D RM by sucrose gradient flotation (see basic protocol, steps 5 to 7). Obtain and centrifuge the D RM from the interface (see basic scheme, steps 8b to IOb), using the S W 28 rotor instead of the S W 41 rotor and increasing the volume proportionally.

3. I ml of methanol is added to the D RM precipitate, which is vigorously shaken until the precipitate suspends and is transferred to a 15 ml glass tube with a temperolined lid. Wash twice with I ml of methanol, combining each wash solution into the original tube. Add 3 ml of chlorine.
Chloroform, followed by 6 ml of a 1 : 1 (WV) mixture of methanol and chloroform.

4 . Apply a high intensity bath type sonicator (recommended) or a standard sonicator for a few seconds. Incubate overnight at room temperature with shaking. Filter under vacuum through a 24m m Whatman G F /C glass fiber filter into a 125m l side arm flask.

5 . Rinse the filter with IOml I : I (V7 V ) methanol and chloroform solution. Pour the solution into a 125 m l flat-bottomed flask with a ground glass neck suitable for rotary evaporator and dry with rotary evaporator. Dissolve the lipids in 5 ml of Solvent A.

6 . Using a glass rod, tuck a small roll of glass wool into the tip of an IO m l wide-mouth glass pipette. Attach the column to a ring stand connection or clamp it in place. Fill the pipette and glass wool with 2 ml of silanization reagent. Rinse with 5 ml of methanol and make sure that the glass wool does not get on the inside wall of the pipette.

7 . Using an IOm l silanized pipette, add enough DEAE dextran gel to the column to form a 2m l column bed. Fill the column with 10 times the column volume of Solvent A. Use a pasteurized pipette to bring the dissolved lipids up the column. Fill the column with 10x column volume of

16. Solubilize the lipids with I : I (V/V) chloroform/methanol. Adjust the concentration of each sample to an expected sample volume of 5 to 20 ul (1 to 7/xg of each lipid). Test several concentrations to ensure that one is within the range of the standard curve.

17. The first sample is loaded with a modified 10M1 Hamilton syringe and a series of very small droplets spreading to 5 m m wide are dispensed between two points on the start line. Allow the solvent to dry briefly and then proceed to dispense smaller droplets on the previous sample, continuing to dispense until the desired sample volume is reached.

1 8 . Sample the remainder of the sample and select a suitable neutral or acidic lipid control, sample 5 ul (1 to 7ug of lipid), and dry the plate at room temperature for 5m in . 临用前在带磨口玻璃塞的 IOOm l 量 筒 中 配 制 70. 8m l 溶 剂 I :4 2 m l 氯仿、 1 8m l 甲醇、 7 . 2 m l acetic acid, 2 . 4 m l formic acid and 1 . 2 m l of distilled water.

19. Add the solvent and absorbent paper to the TLC cylinder, saturate the absorbent paper, immediately cover with a slide and press with a heavy object (e.g., two 4L water jars filled with the liquid) to prevent evaporation. Place the sample-spotted TLC plate in the TLC cylinder so that the starting position is at the bottom of the cylinder, cover immediately and press down with a heavy object.

20. When the solvent front is 6 cm from the bottom of the plate (at the position marked with a pencil), remove the plate from the tank and let it air dry for 15 to 30 min. If necessary, it can also be dried with cold air from a hairdryer. During the evaporation of the solvent, 102 ml of Solvent System II: 65 ml of hexyl ether, 35 ml of isopropyl ether, and 2 ml of acetic acid can be prepared simultaneously in an IOOml cylinder with a ground glass stopper.

21. Add the solvent and blotting paper to another T L C cylinder, saturate the blotting paper and cover immediately to prevent evaporation. When the first solvent has evaporated, place the T L C plate with the sample spotted on it in the T L C cylinder. Swim until the solvent front reaches the top of the plate (10 c m ). Remove the plate from the vat and air dry for 15-30 min. Discard the used solvent and air dry the blotting paper thoroughly for reuse (can be reused several times).

22. Wet the plate by immersing it in a lipid-based developing reagent or by spraying the liquid on the plate in a fume hood. Stand it up and drain the liquid until the gloss of the plate fades.

23. The plate is dried in a preheated 100°C oven for 1 to 2m in before it changes color and then placed in another 180°C oven on a 0.5 to I.O. thick aluminum block for 5 to 10 minutes. O cm thick aluminum block in another 180 °C oven for 5 to 100 m in until the color is well developed but the background color has not yet appeared, then remove and allow to cool for use. If storage is required (up to 24 h), the old TLC plates should be used for storage.
If storage is required (up to 24 h), the plate should be covered with an old TLC plate (from which the silica gel has been removed), wrapped in plastic film, and stored at 1-20°C.

2 4 . The ribbons were scanned by densitometry within 24 h. A standard curve was drawn using neutral or acidic lipid standards, and the samples were measured against the appropriate standard curve.

SUPPLEMENTARY OPTIONS 4 Preparation of DEAE dextran gels MATERIALS

D E A E Dextran Gel A -25

lmol/L N a O H

0.5 m o l / L acetic acid: dilute 57.5 m l of glacial acetic acid in 2 L of water

Methanol

Solvent A : 30 : 60 : 8 (V/V/V) Chloroform/Methanol/Water

4L side arm flask

Brinell funnel with filter paper (18.5 cm diameter)

250 ml reagent bottle (with ground glass stopper or Teflon lined screw cap)

Supplementary program 5 C1 8 Preparation of reversed-phase chromatographic columns Materials

C 1 8 Reversed-phase Grains Cinnamon (Millipore)

I : 2 ( V A O Chloroform/Methanol

Methanol

glass wool

5 ml - disposable glass pipettes

125 ml side arm flask with single stopper

I ml disposable glass flasks

1 . Fill a 5-mL disposable glass pipette with a small amount of glass wool. Pour in the reverse-phase C18 granular silica to form a 1.5-ml column bed. Then place a small piece of glass wool on top.

2 . Place the pipette on a 125 ml side arm flask with a single stopper and attach a vacuum aspirator. Wash with 35 ml of 1:2(VAO) chloroform/methanol, followed by 5 ml of methanol, and finally 10 to 20 ml of water, all under vacuum.

3 . Before each use, repeat the above washing procedure to remove free C 18. Be careful not to add organic solvents containing chloroform directly after adding water. Store dry for up to one year and reuse up to 10 times.

Option 6 Cholesterol Removal with Methyl-P-Cyclodextrin to Destroy Lipid Rafts Additional material

P B S

Pre-warmed serum-free medium with or without 10 m m l/L methyl-P-cyclodextrin (M B C D, Sigma) and lmg/ml B S A

Buffered Salt Solution (BSS, see Appendix 1) with and without 10 mmol/L methyl-p-cyclodextrin and l m g /ml BSA

For adherent cells
L a . Spread the cells in IO cm dishes and wash twice with PBS when the pooled growth reaches 70% to 90%. Add 2.5 ml of pre-warmed serum-free medium containing 10 ml/L methyl cyclodextrin and lmg/ml BSA.

The fullness of the cells has a great influence on the removal of cholesterol, and should be strictly controlled to maintain consistency in different batches of experiments.

2 a. Incubate at 37°C for 15 min. Incubate at 37°C for 15-60 min (optimize incubation time according to Supplementary Scheme 7). Remove the medium and wash twice with serum-free medium. The desired cell function assay can be performed after adding IO ml of serum-free medium.

The oscillation of the petri dish during incubation will strongly affect the removal of cholesterol, which should be strictly controlled to maintain consistency in different batches of experiments.

For non-adherent cells
Ib. Wash cells twice with BSS. Resuspend cells to 2 X ¹⁰⁶ to 4 X ¹⁰⁶ cells/ml with BSS containing 10 mMol/L methyl-(3-cyclodextrin and lmg/ml BSA. Add 2.5 ml of pre-warmed serum-free medium containing 100 mol/L ^methyl-β-cyclodextrin and I mg/ml BSA. Incubate at 37°C for 15-60 min (optimize incubation time according to Supplementary Scheme 7).

2b. Incubate at room temperature, centrifuge at 225 g for 5 to lO min. wash twice with BSA. Add IO ml of serum-free medium and the desired cell function assay can be performed.

Kinetics of cholesterol removal by Methyl-P-Cyclodextrin (MBCD) treatment Materials

60~lOOCi/mmol [1,2,6, ^7-3 H (N )] cholesterol ([ ³ H] cholesterol)

Serum-containing medium

Cells

PBS
Serum-free medium with 1 % (m /V) B S A

B S S, optional

Serum-free medium with 10 mmol/L M B C D (Sigma)

PBS with 10 % (VAO Triton X-100)

60 m m Tissue Culture Dish

Liquid flash meter

1 . Add l ~ 2MCi/m l [ ³ H ] cholesterol to the serum-containing medium under sterile conditions to maintain a solvent concentration of 0 . 1 %. Incubate at 37°C for 12 to 24 h to equilibrate cholesterol with serum lipoproteins. Prepare 5 60-mm dishes of adherent cells, which are expected to reach confluence and grow to 70% to 90% after 2d, or non-adherent cells, which are expected to reach confluence and grow to 70% to 90% after 2d.
The cell volume is expected to reach I O X l o ⁶ cells after 2 d.

2 . Add normal culture volume of [ ³ H]cholesterol-containing medium, incubate for 2 d, remove medium, and wash twice with PBS.

3 . Transfer the cells to new 60 m m dishes to avoid [ ³ H]cholesterol bound to the plastic dishes from interfering with the results. For adherent cells, trypsin digestion is performed and the cells are resuspended in serum-containing medium in new dishes to promote adherence. After wall attachment, wash twice with PBS and replace with serum-free medium containing 1 % BSA. For non-adherent cells, resuspend in serum-free medium containing 1 % BSA. Incubate overnight at 37°C.

4 . Take 2 X ¹⁰⁶ suspended cells or an adherent cell that has grown to 70%~90% confluence in a dish, wash it twice with PBS or BSS, and replace it with I ml of supernatant-free medium containing 10 mmol/L MBCD (may contain lmg/ml BSA). The control group was replaced with the same medium without MBCD. Incubate at 37°C for 10 min, 20 min, 30mn or 60 min.

5 . Remove and retain the medium (adherent cells) or cellular starch (non-adherent cells) and wash with PBS or BSS. For poorly adherent cells, transfer the medium to a small 1.5 ml centrifuge tube and centrifuge at maximum speed for 5 m i n to settle the cells and remove floating cells or large cellular debris. Pipette the supernatant into a new centrifuge tube.

6 . Lyses monolayers of adherent cells or centrifuged suspension cells with PBS containing 10 % (WV) TritonX-IOO for 20 min at room temperature. centrifuge at maximum speed for 1 min and transfer the supernatant to a new centrifuge tube.

7 . Bring the remaining clarified medium to a concentration of 1% Triton X-10 with 10% Triton X-10 in PBS. Add 1/10 volume of PBS to the clarified cell lysate. Count the radioactivity of a double 100 W of clarified cell lysate or medium using a liquid flash meter. Determine the total percentage of medium counted in each trial.

Auxiliary Option 8 Cellular Cholesterol Removal Using MBCD-Cholesterol Complexes Materials

Cholesterol
I : 1 chloroform/methanol
Methyl-P-cyclodextrin (M B C D ; Sigma)
Serum free medium
V PBS or BSS
10 to 15 ml glass tubes with Teflon-lined screw caps

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