Revised 04/2007
DBL
Lactate Dehydrogenase (LDH) Assay
LDH assays can be performed by assessing LDH released into the media as a marker of dead cells or
performing lysis LDH as a marker of remaining live cells.
Neuronal enriched cultures – you can do either Lysis LDH or (“regular”) LDH. If we say LDH assay in the
lab, we mean the non-lysis form. This protocol is described below the regular LDH assay.
Mixed cultures – must use LDH assay. Lysis LDH would cause release of LDH from astrocytes, and we
are only interested in measuring neuronal cell death.
1.
Prepare Lactate Dehydrogenase Assay Mixture fresh for each experiment: Mix equal amounts of
Lactate Dehydrogenase Assay Substrate, Assay Dye, and Enzyme in order to have 20 µL for each
well that will be measured plus 10% extra for error . Enzyme aliquots are in the -20ºC freezer and
are in 400 µL and 750 µL volumes. 400 µL is sufficient for one plate; 750 µL is sufficient for 2
(24) well plates. The Substrate and Dye are in the tissue culture refrigerator. The enzyme must be
kept in the dark, and the reagents are added in the dark, with the hood light off.
Calculation example: 750 µL enzyme, 750 µL dye and 750 µL substrate for a total volume of 2.25
mL.
1
2.
In a 96 well clear plate, fill all wells in column A with 40 µL MEM/BSA/Hepes (+/- N2 if
appropriate) as blank.
3.
Pipette 40 µL of each sample from your toxicity experiment in duplicate into the 96 well plate. For
example, the samples from a 24 well plate could be loaded as follows:
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.
Revised 04/2007
DBL
4.
Using a repeater, add 20 µL of the substrate/dye/enzyme mix to each well. (Pop any bubbles with
a needle/forceps)
5.
Cover the plate with a paper towel and bring it to the plate reader outside the cold room.
6.
Open the Magellan program on the desktop. Use the manual setting to shake the plate for 5
seconds, and then store it in a dark location (inside the shelf under the laptop) for 20-30 minutes at
room temperature.
7.
Use the Magellan program on the plate reader outside the cold room to measure absorbance at a
wavelength of 492 nm. Print your data and save results.
8.
Plot your data subtracting the column A as a blank from all data points. See Excel spreadsheet of
LDH assays for example of calculations and for use as templates for entering data.
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.
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.
Following these steps for lysis, the protocol is the same as the “regular” LDH assay, beginning at step one.
The single difference in the 96 well plate comes from PBS/lysis buffer being used for blanks, instead of
MEM/BSA/Hepes.
Notes:
Total Assay Time: 15 minutes to 1 hour
Purpose: To assess cell viability
Equipment: Repeater pipette, spectrophotometer, Sigma Tox-7 Kit (“In Vitro Toxicology Assay Kit, lactic
dehydrogenase based”), 1 mL pipette, 100 µL