Assessment of Plasma Membrane Fatty Acid Composition and Fluidity Using Imaging Flow Cytometry

Abstract

Phospholipid fatty acid (FA) composition influences the biophysical properties of the plasma membrane and plays an important role in cellular signaling. Our previous work has demonstrated that plasma membrane fatty acid composition is an important determinant of oncogenic Ras signaling and that dietary (exogenous) modulation of membrane composition may underlie the chemoprotective benefits of long chain n-3 polyunsaturated fatty acids (PUFA). In this chapter, we describe in vitro methods to modulate membrane phospholipid fatty acid composition of cultured cells using fatty acids complexed to bovine serum albumin (BSA). Furthermore, we describe a method to quantify the biophysical properties of plasma membranes in live cells using Di-4-ANEPPDHQ (Di4) and image-based flow cytometry.

1. Introduction

The plasma membrane (PM) is a dynamic cellular structure composed of a myriad of lipids and proteins [1]. The coalescence of these membrane components into structured domains regulates many cellular signals, including Ras [2]. Therefore, disruption of these structured domains may serve as a complementary strategy to inhibit aberrant Ras signaling [3]. Recently, we demonstrated that membrane enrichment with long-chain n-3 polyunsaturated fatty acids (n-3 PUFA), e.g., eicosapentaenoic acid (EPA, 20:5 ^{Δ5,8,11,14,17}) and docosahexaenoic acid (DHA, 22:6 ^{Δ4,7,10,13,16,19}), attenuates oncogenic signaling by altering KRas proteolipid composition [4].

The physiological delivery of fatty acids to mammalian cells occurs via non-esterified fatty acids bound to serum albumin or by their incorporation into circulating lipoproteins. Thus, the delivery of PUFA by bovine serum albumin (BSA) provides an efficient strategy to manipulate membrane composition.

The ability to assess the biophysical properties of the plasma membrane is of great interest since plasma membrane rigidity is linked to plasma membrane receptor function and downstream cellular signaling [5–7]. A commonly used method to assess membrane order involves confocal-based spectral ratiometric imaging of polarity-sensitive membrane dyes, e.g., Laurdan and Di-4-ANEPPDHQ [8, 9]. This technique has been recently extended to flow cytometry-based applications [10, 11]. Here, we describe methods that combine imaging and flow cytometry to assess membrane order [5, 12, 13]. This application has the advantage of rapid single cell analysis similar to flow cytometry while also providing images which can be masked and thresholded to determine organelle-specific mem brane order.

Dietary n-3 PUFA play an under-appreciated role in human health and disease as modulators of membrane structure and function [12–14]. To elucidate the interrelationships between long-chain fatty acids and membrane rigidification, we describe protocols to modulate membrane fatty acid composition via the delivery of albumin-complexed fatty acids to cells in culture. Furthermore, we describe a quantitative method (imaging flow cytometry) to determine the biophysical impact of fatty acid incorporation on plasma membrane order.

2. Materials

2.1. Delivery of Fatty Acids

1. Baked spatulas, 250 mL beaker, and 2 mL glass conical V-bottom vials.

2. Bovine Serum Albumin Fraction V, heat shock, fatty acid-free (Sigma, 3117057001), MW: 68,000 g/mol.

3. DHA [52.56 mg/mL in EtOH], (Nu-Chek Prep, U-84-A) MW: 328.57 g/mol.

4. 0.05 M Na₂CO₃ buffer: 0.053 g Na₂CO₃ in 10 mL sterile H₂O

5. 15% BSA solution: Add 20 mL of RPMI 1640 medium into 50 mL beaker. Gently layer 3 g of FA-free BSA onto the surface of the medium. Do not stir, let the BSA powder slowly dissolve into the medium.

2.2. Determining Membrane Order

1. We are providing details of our set-up using an Amnis^® FlowSight^®. However, an equivalent image-based flow cytometer could be used as long as it has the following specifications: A 488 nm laser and capable of collecting emissions 480–560 nm (ordered) and 640–745 (disordered) to analyze Di-4-ANEPPDHQ (see Note 1).

2. Live Cell Imaging Solution (Thermo Fisher, A14291DJ).

3. Di-4-ANEPPDHQ (Thermo Fisher, D36802).

4. 10 mM MβCD cholesterol depletion buffer: 0.066 g MβCD (Sigma, C4555) into 5 ml. RPMI 1640.

3. Methods

3.1. Preparation of BSA-FA

1. Dissolve the fatty acids in 100% ethanol (see Note 2).

2. Add 5 mg of FA into a 2 mL glass vial.

3. Carefully dry down the FA under a stream of N₂.

4. After drying down the FA, add I mL of 0.05 M Na₂CO₃ to the vial. Flush the vial with N₂ gas. Vortex for 30 s. Let the vials sit at room temperature (RT) for 1 h. During the 1 h incubation, vortex the vials every 10 min to aid the dissolution of the FA.

5. Calculate the materials needed to generate a 2.5 mM FA-BSA complex at the FA:BSA (3:1) molar ratio (2.5 mM refer to FA concentration).

   Example: DHA (MW: 328.57 g/mol): 5 mg in 1 mL 0.05 M Na₂CO₃

   BSA (MW: 68,000 g/mol): 15% solution

6. Calculate the volume of 15% BSA solution needed for 5 mg DHA to obtain FA:BSA 3:1 molar ratio.

   Example:

   $$\begin{array}{l}\frac{0.005\text{g}\text{DHA}}{328.57\text{g}/\text{mol}(\text{DHAMW})}\times \frac{1}{3}\times 68,000\text{g}/\text{mol}(\text{BSAMW})\\ \times \frac{100}{15}\\ =2.3\text{mL}\end{array}$$

7. Calculate the total volume of solution needed for 5 mg DHA to make 2.5 mM DHA-BSA complex.

   Example:

   $$\frac{5\text{mg}\text{DHA}}{328.57\text{g}/\text{mol}(\text{DHAMW})\times 2.5\text{mM}}=0.006087\text{L}=6.09\text{mL}$$

8. Calculate the volume of basal medium needed for making the 2.5 mM DHA-BSA complex.

   Example:

   6.09 mL (total volume) − 1 mL (FA in 0.05 M Na₂CO₃) − 2.3 ml. (15% BSA) 2.79 ml.

   DHA	15% BSA	Medium	2.5 mM DHA-BSA
   	1 mL	2.3 mL	2.79 mL	6.09 mL (total volume)

9. It is difficult to completely retrieve the 1 mL FA-Na₂CO₃ from the glass vial. Therefore, take only 95% of the solution to make the DHA-BSA complex.

   Example: 0.95 mL of DHA + 2.19 mL of 15% BSA + 2.65 mL of medium.

10. Add the following mL of FA-Na₂CO₃, 15% BSA, and medium to 15 mL conical tube for respective FA.

    Ex:	FA-Na2CO3	15% BSA	Medium	(FA-BSA)
    	0.95 mL	2.19 mL	2.65 mL	(5.79) mL

11. Flush the tubes with N₂. Shake the tubes on a belly dancer shaker for at least 0.5 h.

12. Filter sterilize using a 0.2μm low protein binding syringe filter and aliquot the FA-BSA complex under a sterile hood (see Note 3).

13. Store at −20 °C for up to a month.

3.2. Treatment of Cells

1. Seed 5 × 10⁴ young adult mouse colonic (YAMC) cells per well of a 24-well plate. Remember to have an extra well to use as a cholesterol depletion control (see Note 4).

2. Incubate cells with desired concentration of FA-BSA for 24 h (see Note 5).

3.3. Trypsinization and Labeling with Di-4-ANEPPDHQ

1. To deplete cholesterol, remove media and replace with RPMI media containing 10 mM methyl-β-cyclodextrin (MβCD) for 30 min at 33 °C (see Note 6).

2. For a 24-well plate, aspirate media and rinse with 1 mL DPBS. Add 250μL of 0.05% trypsin-EDTA for 3–5 min.

3. After 3–5 min, add 1 mL of media to stop the trypsinization. Transfer the cell suspension into a 2 mL tube. Centrifuge cells at 200 × g for 5 min to pellet cells.

4. After centrifugation, aspirate media and resuspend cells in 50μL of live cell imaging solution (LCIS) or other imaging media. Keep cells on ice.

5. When ready to image, add 4μL of 10μM Di4 in LCIS media to an aliquot of 36μL of cells in LCIS (Final Di4 concentration: 1 μM). Pipette up and down gently, to mix and avoid generating bubbles.

6. Immediately run sample on the FlowSight (see Note 7).

3.4. Imaging

1. Perform the startup and calibration of the FlowSight according to manufacturer’s recommendation. Takes ~30 min.

2. Set a gate to collect whole cells as in Fig. 1 (see Note 8).

3. Adjust the power of the 488 nm laser to minimize saturated max pixels.

4. Collect at least 5000 events (see Note 9).

Fig. 1.

Representative FlowSight gating strategy

3.5. Analysis

1. Analysis is performed using commercial software associated with the flow cytometer. On our machine, we use Amnis IDEAS^® (Version 6.2).

2. For every cell, the mean intensity of CH02 (ordered, O) and CH05 (disordered, D) is used to generate a generalized polarization (GP) value by applying the equation below [9].

   $$\text{GP}=\frac{I_O-I_D}{I_O+I_D}$$

   where I_O and I_D are the intensities of ordered channel and disordered channel, respectively. The GP value ranges from +1 to −l, where higher values indicate increased membrane order. The analysis template used is available upon request.

3. Cholesterol depletion by MβCD can be used as a control to reduce membrane order (see Note 5). An example of representative data is shown in Fig. 2.

Fig. 2.

DHA enrichment and cholesterol depletion by MβCD reduce membrane order. (a) Representative bright field (BF), ordered (O) and disordered (D) channel images of YAMC cells. Plasma membrane, whole cell, or intracellular membrane masks overlaid in blue highlight. When images are acquired quickly (<10 min), most of the fluorescence signal is localized to the plasma membrane. Under these conditions, the whole cell mask is sufficient for plasma membrane GP analysis. Quantitative (b) raw and (c) normalized data from YAMC cells untreated or incubated with DHA-BSA (50μM) for 24 h or MβCD (10 mM) for 30 min prior to determination of membrane order with Di-4-ANEPPDHQ by image-based flow cytometry. Data represents mean GP ± s.d. from whole individual cell mask: untreated (3382 cells), DHA (2617 cells), and MβCD (2625 cells). Statistical significance between treatments as indicated by uncommon letters (p < 0.0001) was examined using one-way ANOVA and uncorrected Fisher LSD tests