|Quantity (with Count)
|1 x 1 ea
|1 x 500 µl
|LDH Positive Control
|1 x 1 ea
|Lactic Acid (100X)
|1 x 500 µl
|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.
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