Detection of Necrosis by Release of Lactate Dehydrogenase (LDH) Activity

Summary

Apoptosis and necrosis are two major forms of cell death observed in normal and disease pathologies. Although there are many assays for detection of apoptosis, relatively few assays are available for measuring necrosis. A key signature for necrotic cells is the permeabilization of plasma membrane. This event can be quantified in tissue culture settings by measuring the release of the enzyme lactate dehydrogenase (LDH). When combined with other methods, measuring LDH release is a useful method for detection of necrosis. In this chapter, we describe the step-by-step procedure for detection of LDH release from necrotic cells using a microtiter plate based colorimetric absorbance assay.

1. Introduction

Necrosis is a type of cell death that is morphologically characterized by swelling and rupture of intracellular organelles, eventually leading to the breakdown of the plasma membrane (1–3). Plasma membrane leakage from necrotic cells causes the release of intracellular contents into extracellular milieu. Therefore, necrotic cell death evokes inflammatory responses and is closely associated with inflammatory diseases (4). In contrast to apoptosis, necrosis was once considered as a passive and accidental form of cell death (5). However, several recent studies have clearly established that necrosis can be a programmed event (6–8). Death cytokines in the tumor necrosis factor (TNF) family are potent triggers of necrosis. In this chapter, we describe a convenient, non-radioactive and high-throughput method to detect necrosis, including secondary necrosis, based on the leakage of a cytoplasmic enzyme, lactate dehydrogenase (LDH), from the damaged cells.

Historically, necrotic cell death is evaluated by determining damage of the plasma membrane. The cytotoxicity assays for measuring necrosis are principally divided into two categories: those that are based on differential uptake of DNA binding dyes that do not traverse the plasma membrane of living cells, such as propidium iodide; and those that are based on the leakage of intracellular molecules through impaired plasma membrane. LDH is a soluble cytoplasmic enzyme that is present in almost all cells and is released into extracellular space when the plasma membrane is damaged (9). To detect the leakage of LDH into cell culture medium, a tetrazolium salt is used in this assay. In the first step, LDH produces reduced nicotinamide adenine dinucleotide (NADH) when it catalyzes the oxidation of lactate to pyruvate. In the second step, a tetrazolium salt is converted to a colored formazan product using newly synthesized NADH in the presence of an electron acceptor (10). The amount of formazan product can be colorimetrically quantified by standard spectroscopy. Because of the linearity of the assay, it can be used to enumerate the percentage of necrotic cells in a sample (see Note 1).

2. Materials

2.1. Equipment

1. Microtiter plate reader (FluoStar Optima, BMG LABTECH Inc, Ortenberg, Germany).

2. Centrifuge with plate adaptors (Sorvall Legend RT, DJB Labcare Ltd, Buckinghamshire, England).

3. Multichannel pipettes (Brand, Wertheim, Germany) and repeat pipettor (Eppendorf, Hamburg, Germany).

2.2. Reagents and Supplies

1. Flat bottom or U-bottom 96-well microtiter plate.

2. RPMI 1640, Dulbecco’s Modified Eagle Medium, or any other media depending on the cell type used (Invitrogen, CA, USA) (see Notes 2–4).

3. Fetal bovine serum (Any vendors) (see Note 5).

4. Tris Buffer, 200 mM, pH 8.0. Dissolve 24.2 g Tris base in 1 L milli-Q water. Adjust pH to 8.0 with hydrochloric acid. Sterilize by autoclave.

5. Prepare the 2X LDH assay buffer as follows: Dissolve 223 mg INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride), 57 mg PMS (N-methylphenazonium methyl sulfate) (see Note 6), 575 mg NAD (nicotinamide adenine dinucleotide), and 3.2 g lactic acid in 480 mL 200 mM Tris buffer solution, pH 8.0 (see Notes 7–8). Store the 2X LDH assay buffer in small aliquots at −20°C (see Notes 9–10).

6. 10X Lysis solution: 9% Triton X-100 (Thermo Fisher Scientific, NH, USA). Dissolve 9 mL Triton X-100 with 91 mL milli-Q water.

7. Stop solution: 1 M acetic acid (Thermo Fisher Scientific, NH, USA) (see Note 11).

3. Methods

All the reactions should be performed at room temperature (22 – 25°C). Warm all the components to 22 – 25°C before starting the experiment.

3.1 Cell culture preparation (see Note 12)

1. Seed cells in a 96-well flat bottom microtiter plate at a density of 1 × 10⁴ – 5 × 10⁴ cells/well in 100 μL of culture medium. Samples in each experimental group should be prepared in triplicate wells (see Notes 13–14).

2. On the same plate, prepare the following controls in triplicates.

   • Maximum LDH release – Seed the same number of cells in these wells as in step 3.1.1.

   • Medium alone – these wells contain only medium but no cells.

   • Volume Correction Control – these wells contain only medium but no cells.

3. For adherent cells, allow the cells to incubate for 12 – 16 hours prior to treatment. For suspension cultures, treatment can be initiated when seeding of cells is completed.

4. Culture the cells for the required amount of time in a humidified 37°C incubator equilibrated with 5% CO₂.

3.2 Harvest Supernatants

1. To the maximum LDH release wells and the volume correction control wells, add 10 μL of 10X lysis solution.

2. Return the plate to a humidified 37°C, 5% CO₂ incubator for 45 minutes.

3. Centrifuge the microtiter plate in a centrifuge at 1,500 – 2,000 rpm for 5 minutes (see Note 15).

4. With a multi-channel pipette, collect 50 μL of culture supernatant from each well and transfer it to a new microtiter plate (see Notes 16–18). Be careful not to transfer any cell materials.

3.3 LDH measurement

1. Add 50 μl the reconstituted 2X LDH assay buffer to the supernatants. Mix the reagent by gentle shaking for 30 seconds (see Notes 18–20).

2. Protect the assay plate from light. Incubate the plate at room temperature (22 – 25°C) for 10 –30 minutes (see Note 21).

3. Add 50 μL of Stop Solution to the wells. Mix the reagent by shaking for 30 seconds (see Note 22).

4. Measure absorbance between 490 – 520 nm (see Note 23).

3.4 Data Analysis

1. The reading from the untreated and test wells is subtracted with the reading from the medium alone control wells. This is the corrected reading for the untreated or test well.

2. Subtract the reading of Volume correction control wells from the reading of the Maximum LDH release wells. This is the corrected maximum release reading.

3. Calculate the percentage of cytotoxicity using the following formula:

   $$\begin{array}{l}\%\text{Cytotoxicity}=100\times (\text{Corrected}\text{reading}\text{from}\text{Test}\text{well}-\text{corrected}\text{reading}\text{from}\text{untreated}\text{well})/(\text{Corrected}\\ \text{maximum}\text{LDH}\text{release}\text{control}-\text{corrected}\text{reading}\text{from}\text{untreated}\text{well})\end{array}$$