Cellular Copper Death Assay

Summary

Copper-dependent cell death is a new form of cell death, defined as a non-apoptotic cell death pathway. In healthy cells, copper ions enter the cell through copper transporter proteins, and also participate in oxidation-phosphorylation reactions through oxidoreductases and other enzymes in the mitochondria to complete the cellular metabolic process.

However, when the concentration of copper ions in the cell is too high, or when the copper transporter protein or oxidoreductase is missing, excessive copper ions will accumulate in the cell, inducing oxidative stress, damage to the cell membrane DNA, thus affecting the normal function of the cell, and ultimately leading to cell death.

Principle

Copper binds directly to the lipoylated components of the tricarboxylic acid (TCA) cycle. Aggregation of these copper-bound lipoylated mitochondrial proteins and degradation of Fe-S cluster proteins then triggers protein hydrotoxicity stress, ultimately leading to cell death.

Operation method

Detection of cellular copper death

Principle

Copper binds directly to the lipoylated components of the tricarboxylic acid (TCA) cycle. Aggregation of these copper-bound lipoylated mitochondrial proteins and degradation of Fe-S cluster proteins then triggers protein hydrotoxicity stress, ultimately leading to cell death.

Materials and Instruments

Reagents: DMEM medium, fetal bovine serum, penicillin-streptomycin, Disulfiram (DSF), 0.25% trypsin (with EDTA), phosphate buffer solution (PBS), CuCl2 solution, antibodies (FDX1, DLAT, DLST, LIAS, caspase 3, caspase 9, GAPDH and HRP), DAPI dye, SDS-PAGE gel

Move

1, the cells were placed in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin for growth. 2, the cells were digested with 0.25% trypsin and then collected;

2, take the cells in the logarithmic growth phase, collect the cells after digestion with 0.25% trypsin, and resuspend them with PBS to adjust the cell concentration to 2 × ¹⁰³ cells/well and inoculate them into 96-well plates;

3. Add 1 µM _CuCl2 solution into the culture 30 minutes in advance, then add 0.05~1.6 µM DSF-treated cells into each well, and set up a blank control group (i.e., DSF-treated group without _CuCl2 ), and place them in 37 ℃, 5% _CO2 incubator;

4. After 16~24 hours of cell treatment, add 0.05~1.6 μM DSF three times, and then measure the cell viability by the kit after 72 hours, and observe the cells under the microscope;

5. The cells of the treatment group in step 3 were treated with DSF for 2 hours and then washed with PBS, resuspended with 10% formalin and incubated at room temperature for fixation;

6. Incubate the cells with FDX1 antibody as primary antibody, add fluorescently labeled secondary antibody after washing away the excess primary antibody with PBS, then wash the cells with PBS buffer, and stain the samples with nuclear staining agent (DAPI) for observation under the microscope;

7, the same DSF-treated cells after 2 hours of protease inhibitor lysis, elution of proteins to the SDS-PAGE gel for electrophoretic separation, and then transferred to a polyacrylamide film, the transmembrane closed and added primary antibody (FDX1, DLAT, DLST, LIAS, caspase 3, caspase 9, GAPDH) at 4 ℃ incubation overnight, and then add the secondary antibody HRP at room temperature, and then add the secondary antibody HRP at room temperature, and then add the secondary antibody HRP at room temperature. The membrane was incubated overnight at 4 ℃ with primary antibody (FDX1, DLAT, DLST, LIAS, caspase 3, caspase 9, GAPDH), and then incubated with secondary antibody HRP for 2 hours at room temperature on a shaker.

Caveat

(1) Cell culture conditions need to be strictly controlled, with temperature, humidity, CO2concentration, and medium formulation to ensure the reproducibility of the experimental results;(2) Copper ion concentration and treatment time should be controlled, excessive concentration and prolonged treatment will lead to unnecessary toxicity and interference, affecting the experimental results；

（3） Use electric pulse when treating with copper ion carrier drug DSF to avoid inducing apoptosis and generating oxidative stress in cancer cells due to the drug；

（4） Avoid changes in cell morphology and structure caused by additional pressure or other factors during cell fixation, which may affect the experimental results. The selection of staining reagents should be optimized and adjusted based on specific situations, and the compatibility of staining reagents with cell samples should be noted；

（5） In the protein detection index, focus on observing the changes of Fe-S protein, while the content of apoptosis-related proteins will not change, if there are obvious changes in apoptosis-related proteins, we should consider whether there is any error in the operation method during the experimental process.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/