|37291S||1 Kit (480 assays)||
|Product Includes||Quantity (with Count)|
|Diaphorase||1 x 1 ea|
|INT (100X)||1 x 500 µl|
|LDH Positive Control||1 x 1 ea|
|Lactic Acid (100X)||1 x 500 µl|
|NAD+ (100X)||1 x 500 µl|
|Triton™ X-100 (10%)||1 x 10 ml|
|Cell-Based Assay Buffer Tablet||1 x 1 ea|
To make 10ml of LDH Reaction Solution, sufficient for use on one 96-well plate, add 100ul of the following to 9.6ml of assay buffer:
Any leftover LDH Reaction Solution should be discarded after use, as it is not stable. If less than a full 96-well plate is to be used in an experiment, adjust the volumes of each of the reactants accordingly. Store remaining INT (100X) at -20°C. Store NAD+ (100X) and lactic acid (100X) at 4°C.
Note: Serum used to supplement growth medium (fetal calf serum, etc.) contains LDH that will react with the LDH Reaction Solution and induce a “background” color change (A490), even in the absence of cell death. The higher the percentage of serum in the medium, the higher the background signal will be. There are two solutions to this background problem; grow the cells in the absence of serum, or subtract the background signal from all wells prior to calculation of % cytotoxicity. Removal of serum from a growth medium can have negative impact on overall cell viability, so this may not be an option for all cell types. Subtraction of the background signal is easier, requiring simply the addition of wells to the assay that contain medium-only, without added cells. The LDH A490 signal resulting from the medium-only controls can be subtracted from all test wells after reading the plate.
Note: Different cell types contain different amounts of LDH. For cells with high LDH levels, fewer cells per well will be required to produce a strong A490 value than for cells with relatively low LDH levels. Therefore, we recommend performing an initial titration experiment to determine the optimal number of cells per well of the target cell you plan to use.
Protocol Id: 2504
Cell death can occur either by apoptosis, a highly regulated biochemical pathway involving signal transduction cascades, or by necrosis. Necrosis is accompanied by mitochondrial swelling and increased plasma membrane permeability, while apoptosis involves an articulated breakdown of the cell into membrane-bound apoptotic bodies (1). There are a number of assays that are designed to measure cytotoxicity and cell death, independent of mechanism. Most of these assays assess cell viability by measuring plasma membrane permeability (2).
Lactate dehydrogenase (LDH) is a stable, soluble enzyme located in the cytosol of many different cell types. The enzyme is released into the surrounding culture medium upon plasma membrane damage. LDH activity in the culture medium can, therefore, be used as a reliable indicator of cell membrane integrity, and thus a measurement of cytotoxicity.
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.