EZQuant™ Cytotoxicity Assay Kit: #CA01

 

Product Description

The EZQuant™ Cytotoxicity Assay Kit is used to assess cell death. It measures the level of LDH released when cells undergo stress or injury from chemicals that are toxic to the cells. The reagent is reduced by NADH that was oxidized by the extracellular LDH. Therefore, the amount of formazan produced is directly proportional to the number of dead cells.

Pair with EZQuant™ Cell Quantifying Kit

The EZQuant™ Cell Quantifying Kit conveniently determines the number of viable cells in cell proliferation and cytotoxicity assays. This product is useful for drug screening and cytotoxicity testing of chemicals. Meanwhile, the EZQuant™ Cytotoxicity Assay Kit determines the number of damaged cells and the degree of cytotoxicity. Combining these two products will allow researchers to fully understand the cell health conditions under various cytotoxic environments.

Features

  • High linearity range
  • Long shelf-life without degradation
    (2 months at 0-5° C)
  • Simple homogenous assay or
    non-homogenous (multiplex) assay possible
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Product Specifications
Product Name Cytotoxicity Assay Kit
Catalog # CA01
Size 500 tests (CA01) / 2000 tests (CA04)
Shipping Room Temperature
Storage and Stability Store at 0-5° C
Quality Control Appearance, blank, and sensitivity tests are performed for each lot. Only lots that pass the following criteria are offered:
Blank Absorbance (460 nm) ≤ 0.700
Sensitivity Absorbance (460 nm) ≥ 1.000
Restricted Use For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Protocol Download
 
  • Homogenous assay
  • Non-homogenous assay
Cell Concentration Optimization
  1. Collect cells and wash them with the assay medium. Prepare cell suspension to 5×105 cells/ml in the assay medium.
  2. Add 100 μl of the assay medium to each well of a flat-bottom 96-well tissue culture plate.
  3. Prepare 2-fold serial dilution of each well in triplicate set of wells for the high-control, low-control and background control (medium only).
    Serial dilution: Add the cell suspension (5×105 cells/ml) to the first well and mix by pipetting. This well contains the maximum number of cells (2.5×104 cells/well). Transfer 100 μl from the first well to the next well, and mix by pipetting. Repeat this procedure.
  4. Incubate the plate at 37° C for an appropriate time in a CO2 incubator.
    Use the same incubation time in the cytotoxicity assay.
  5. Add 10 μl of the lysis buffer to each well of the high control.
  6. Incubate the plate at 37° C for 30 minutes in a CO2 incubator.
  7. Add 100 μl of the working solution to each well. Protect the plate from light and incubate it at the room temperature for 30 minutes.
  8. Add 50 μl of the stop solution to each well.
  9. Measure the absorbance at 490 nm by a microplate reader.
Cytotoxicity Assay
  1. Add 50 μl of cell suspension to each well of a flat-bottom 96-well tissue culture plate.
    For adherent cells: incubate the plate at 37° C overnight in a CO2 incubator to allow the cells to adhere and then replace the assay medium with 50 μl of fresh assay medium.
  2. Add 50 μl of assay medium containing test substance that adjusted to the desired concentration.
      Background control Test substance Low control High control
    Assay medium 100 μl N/A 50 μl 50 μl
    Cell suspension N/A 50 μl 50 μl 50 μl
    Test substance in culture medium N/A 50 μl N/A N/A
    Lysis buffer N/A N/A N/A 10 μl
  3. Incubate the plate at 37° C for an appropriate time period in a CO2 incubator.
  4. Add 10 μl of the lysis buffer to each well of the high control. Incubate the plate at 37° C for 30 minutes in a CO2 incubator.
  5. Add 100 μl of the working solution to each well. Protect the plate from light and incubate it at the room temperature for 30 minutes.
  6. Add 50 μl of the stop solution to each well.
  7. Measure the absorbance at 490 nm by a microplate reader.
Calculate Cytotoxicity

Calculate the average absorbance from each triplicate set of wells and subtract the background control value from each absorbance one. Calculate the percent cytotoxicity by the following equation:
Cytotoxicity (%) = (Test substance - Low control) / (High control - Low control) x 100