CytoSelect™ LDH Cytotoxicity Assay Kit

Catalog No. ABIN2344920

Product Details

Reactivity: Bacteria, Fish, Fusarium, Protozoa, Saccharomyces cerevisiae

Detection Method: Colorimetric

Application: Cytotoxicity Test (CyTox)

Purpose: CytoSelect™ LDH Cytotoxicity Assay Kit provides a colorimetric format for measuring and monitoring cell cytotoxicity.

Brand: CytoSelect™

Sample Type: Cell Samples

Components:

1. LDH Cytotoxicity Assay Reagent : One 10 mL bottle of reagent
2. Triton X-100 Solution : One 10 mL bottle of 10% Triton X-100

Material not included:

1. Cells for measuring cytotoxicity
2. Cytotoxicity mediating compound to be tested
3. Cell culture medium
4. 96-well clear cell culture plates
5. Microtiter plate reader capable measuring absorbance at 450 nm

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• LDH Cytotoxicity Reagent can be used to detect cytotoxicity in bacteria, yeast, fungi, protozoa as well as cultured mammalian and piscine cells
• Cells can be plated and then treated with compounds or agents that affect cell viability
• Colorimetric kit, containing sufficient reagents for the evaluation of 960 assays in 96-well plates

Protocol: The kit contains sufficient reagents for the evaluation of 960 assays in 96-well plates. Cells can be plated and then treated with compounds or agents that affect cell viability. Upon cell death, lactate dehydrogenase (LDH), a soluble enzyme found in the cytoplasm, is released into the growth media. The growth media is then transferred to another plate and the released LDH is then detected with cytotoxicity reagent. In the presence of lactate substrate (included in the LDH Cytotoxicity Reagent) LDH converts lactate to pyruvate and generates nicotinamide adenine dinucleotide (NADH). The WST-1 molecule, also present in the LDH Cytotoxicity Reagent, is converted from WST-1 to the orange formazan form. An increase in cell cytotoxicity is accompanied by increased LDH release and increased colorimetric signal. The assay principles are basic and can be applied to most eukaryotic cell lines, including adherent and non-adherent cells and certain tissues, depending on LDH expression levels. The LDH Cytotoxicity Reagent can be used to detect cytotoxicity in mammalian cells.

Assay Procedure:

Note: Both a negative and a positive control should be run alongside experimental samples. Each experimental sample and control should be assayed in duplicate.

Prepare a cell suspension containing 0.1-1.0 x 10 cells/mL in medium.

Add the cell suspension to a 96-well cell culture plate for each experimental sample, negative and positive control well according to Table 1 below. Include the compound to be tested in experimental wells only. 3 Experimental Negative Positive Sample Control Control Cell Suspension 150 μL 150 μL 150 μL Cytotoxicity-mediating + - - compound to be tested Table

1. Preparation of Experimental and Control Wells.
2. Culture the cells for 24-96 hours at 37 °C and 5 % CO2 in a humidified incubator.
3. Add sterile water or the provided Triton X-100 Solution to each well according to Table 2 below. Experimental Negative Positive Sample Control Control Sterile Water 15 μL 15 μL - Triton X-100 Solution - - 15 μL Table
4. Lysis of Positive Control Cells.
5. Incubate 5-10 minutes at room temperature.
6. Transfer 90 μL of medium from each well to a clean 96-well plate suitable for a plate reader.
7. Add 10 μL of LDH Cytotoxicity Assay Reagent.
8. Incubate plate at 37 °C and 5 % CO2 for 0.5-4 hours.
9. Read OD using 450 nm as the primary wave length. Calculation of Results Positive control wells represent maximal LDH release, while negative control wells represent background LDH release. The OD for negative controls is subtracted from both experimental and positive control OD values, and results are reported as a relative cytotoxicity percentage: OD experimental sample - OD negative control x 100 = % Relative Cytotoxicity OD positive control - OD negative control 4

Restrictions: For Research Use only

Handling

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: RT/-20 °C

Storage Comment: The LDH Cytotoxicity Assay Reagent is a clear, slightly red, ready-to-use solution. Aliquot as needed to avoid repeated freeze-thaw cycles and store at -20°C protected from light. If precipitates or turbidity are observed upon thawing, warm the solution to 37°C for 5-10 minutes and agitate to dissolve the precipitates. Store the Triton X-100 Solution at Room Temperature.

References

Chu, Li, Joshi, Giannopoulos, Hoffman, Madesh, Praticò: "Gamma secretase-activating protein is a substrate for caspase-3: implications for Alzheimer's disease." in: Biological psychiatry, Vol. 77, Issue 8, pp. 720-8, (2015) (PubMed).

Chu, Li, Giannopoulos, Blass, Childers, Abou-Gharbia, Praticò: "Pharmacologic blockade of 12/15-lipoxygenase ameliorates memory deficits, A? and tau neuropathology in the triple-transgenic mice." in: Molecular psychiatry, Vol. 20, Issue 11, pp. 1329-38, (2015) (PubMed).

Li, Chu, Barrero, Merali, Praticò: "Homocysteine exacerbates ?-amyloid pathology, tau pathology, and cognitive deficit in a mouse model of Alzheimer disease with plaques and tangles." in: Annals of neurology, Vol. 75, Issue 6, pp. 851-63, (2014) (PubMed).

Target Details

Background: The measurement and monitoring of cell cytotoxicity is an essential technique in any laboratory focused on cell-based research. This skill allows for the optimization of cell culture conditions. More importantly, the cytotoxic nature of anticancer compounds in toxicology testing, the toxicity of therapeutic chemicals in drug screening, and cell-mediated cytotoxicity can all be assessed through this assay-based approach. Cell cytotoxicity characteristics include loss of cellular metabolic activity or cell membrane integrity. One method for measuring metabolic activity is to incubate the cells with a tetrazolium salt such as MTT, which is cleaved into a colored formazan product by metabolically active cells. Similarly, the green fluorescent dye Calcein AM can measure intracellular esterase activity in proliferating live cells, while dyes such as trypan blue or propidium iodide can enter and stain cells that have lost membrane integrity.