Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Summary

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Abstract

Depletion of the mitochondrial membrane potential (MMP, ΔΨm) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl^- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Introduction

Rh123 is a cationic fluorescence dye, which serves as an indicator for transmembrane potential. Rh123 is capable of penetrating the cell membrane and entering the mitochondrial matrix depending on the potential difference of inside and outside the membrane¹. Apoptosis leads to damage of mitochondrial membrane integrity. The mitochondrion permeability transition pore (MPTP) will open and lead to collapse of the MMP, which in turn results in the release of Rh123 to the outside of the mitochondria. Finally, stronger green fluorescence signal will be detected under fluorescence microscope. It is well documented that depletion of the MMP and elevate....

Protocol

1. Cell Culture and Transfection

1. Cell culture
   Note: HN4, an HNSCC cell line, was derived from patients with HNSCC¹⁴.
   1. Culture HN4 cells in Dulbecco's modified Eagle medium (DMEM, 4.5 g/L glucose) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). Incubate cells at 37 °C with 5% CO₂.
2. Cell transfection
   1. One day before transfection, plate 5 x 10⁵ cells in 2 mL/well of culture medium without antibiotics in 6-well plates.
   2. Dilute 100 pmol CLIC4 siRNA o....

Results

In the present study, Rh123 was applied to detect the MMP. Initially, HN4 cells were cultured for the following fluorescence staining experiments. Tweezers were used to put circular coverslips on the bottom of 6-well plates (Figure 1A). The coverslips were coated with polylysine for 5 min and then the polylysine removed via pipettor (Figure 1B). Then HN4 cells were trypsinized and seeded on the c.......

Discussion

It is well documented that Cl⁻ channels are essential in maintaining the hemostasis of the internal environment and play important roles in cell proliferation and apoptosis^15,16. Therefore, understanding the relationship between ion channel-targeted intervention and apoptosis is of great need and significance to find a better therapeutic approach for various cancers¹⁷. Mitochondria maintain the normal biological state of a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HNSCC cells	ATCC	CRL-3241
Polylysine	Thermo Fisher Scientific	P4981
Specific siRNA for human CLIC4	Biomics	NM_013943	(accession numbers, NM_013943; corresponding to the cDNA sequence 5-GCTGAAGGAGGAGGACAAAGA-3) and scrambled siRNA (5 ACGCGUAACGCGGGAAUUU-3) were designed and obtained from Biomics Company
Lipofectamine 2000 Transfection Reagent	Thermo Fisher Scientific	L3000-015
Opti-MEM I Reduced Serum Medium, GlutaMAX	Thermo Fisher Scientific	51985-042
Rhodamine 123, FluoroPure grede	Thermo Fisher Scientific	R22420
Dulbecco’smodified Eagle medium (DMEM, 4.5 g/L glucose)	Gibco	11965-084
Fetal Bovine Serum, Qualified, Australia Origin	Gibco	10099141
Trypsin-EDTA Solution	Beyotime	C0201
Antibiotic-Antimycotic, 100X	Gibco	15240062
Laser Scanning Confocal Microscopy	Leica Microsystems GmbH	LEICA.SP5-DMI6000-DIC
Nikon Eclipse TE300 Inverted Microscope	Nikon	N/A
Metaflour, V7.5.0.0	Universal Imaging Corporation	N/A
Leica application suite, v2.6.0.7266	Leica Microsystems GmbH	N/A
Microsoft office Excel 2007	Microsoft	N/A
Sigma Plot 12.5	Systat Software	N/A
Attofluor Cell Chamber	Thermo Fisher Scientific	A7816