DL 10/06
Lactate Dehydrogenase Assay (LDH) with lysis
LDH is an enzymatic assay of viability. Lysis LDH is a modification of the assay which allows you to
wash off the “released” LDH then lyse healthy cells to express data as “% viability.” As it lyses all cells, it
cannot by used in mixed cultures which have mostly astrocytes.
1.
2.
3.
4.
5.
6.
1
Prepare PBS/lysis buffer solution, making enough for 500 µl (450 µl of 1X PBS and 50 µl of lysis
buffer for each well on the 24 well plate) and 40 µl for each blank well on the 96 well plate.
Wash cells 2 times with 1X PBS (no lysis buffer) using 1 ml of PBS per well.
Add the PBS/lysis buffer to each well.
Incubate for 45 minutes at 37ºC.
Add 40 µl of PBS/lysis buffer to each blank well (those in the first column) in the clear 96 well
plate. Do not add samples or blanks to row G of the plate.
Add 40 µl of sample to each well, then duplicate the sample in the adjoining well. For example:
Sample Plate (24 wells)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
These samples could be placed in the LDH plate as:
Bl
Bl
Bl
Bl
Bl
Bl
LDH Plate (96 wells)
1 1 7
7 13
2 2 8
8 14
3 3 9
9 15
4 4 10 10 16
5 5 11 11 17
6 6 12 12 18
13
14
15
16
17
18
19
20
21
22
23
24
19
20
21
22
23
24
Bl
Note: No samples are placed in Row G; blanks are loaded into the first column, and following columns
contain the samples in duplicate.
7.
Prepare LDH solution from the substrate, the dye, and the enzyme. Each reagent is added in equal
parts in order to make enough to add 20 µl to each well. The LDH enzyme is kept in 400 and 800
µl in the -20ºC freezer outside the cold room. The enzyme must be kept in the dark (pant pockets),
and the 20 µl of LDH solution are added in the dark, with the hood light off. Use the repeater to
add solution. Pop all bubbles in the sample.
8. Cover the plate with a paper towel and bring it to the plate reader outside the cold room.
9. Using the Magellan program, shake the sample for 5 seconds before getting a preliminary reading.
Then allow the sample to rest for 20-30 minutes in the dark (inside the shelf under the laptop).
10. Without shaking, take measurements, print, and save results.
Supplier: Sigma
Catalog No.: Tox-7
“In Vitro Toxicology Assay Kit, lactic dehydrogenase based”