TITLE: Total lactate dehydrogenase calorimetric enzyme activity measurement
AUTHORS: Renuka Sriram

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lactate Dehydrogenase is a key cytoplasmic enzyme that confers the observed Warburg effect in tumors. Its a tetrameric protein composed of combinations of one or two isomers. This protein catalyzes the reversible reaction </div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dsipbaas7.jpg" /></div><div class = "text-block">This reaction is monitored spectrophotometrically by observing the reduction in absorbance of NADH (which is maximal at 420nm) as pyruvate is converted to lactate. </div><div class = "text-block">This protocol, details the steps involved in such a calorimetric assay of cells and tissues.</div></div>

[STEPS]
?. [Pre-preparation]
Reaction buffer – Tris buffer @ , ,
?. [Pre-preparation]
Measure and add the following: i.	 HCl Trisii.	Tris Baseiii.     of NaCliv.    Use solution to dissolve NADH and add  (10 vials of  each)
1.57 g
0.15 g
1.64 g
20 mg
2 mg
?. [Pre-preparation]
Turn off stir plate and remove stir bar
?. [Pre-preparation]
Store in  freezer as  aliquots (for 12 dilutions of pyruvate). This solution will be stable for upto 6 months.
-20 °C
12 mL
?. [Pre-preparation]
Lysis buffer –  Tris buffer ,  DTT,  EDTA, 1% Triton x-100.
?. [Pre-preparation]
Measure and add the following:i.	 HCl Trisii.	  Tris Base iii.	 EDTAiv.	(or 59.326 uL) of DTTv.	Triton
0.71 g
0.67 g
0.15 g
0.06 g
2 mL
?. [Pre-preparation]
Store as  l aliquots in  C freezer (is stable for up to a year). This is 10 times concentrated, so for the assay add  dH2O.
200 µl
-20 °C
1800 µl
?. [Pre-preparation]
Store  aliquots of sodium pyruvate () in the -freezer. This solution is stable for up to a year when frozen.
500 µl
20 °C
?. [Set-up]
Take from the freezer, one tube each of the pyruvate and reaction buffer and place in  water bath.Thaw the  lysis buffer and add  of dH2O and place on ice
30 °C
1800 µl
?. [Cell preparation]
Trypsinize and collect cells:
?. [Cell preparation]
Cell Counting
?. [Cell preparation]
If using a cell counter, use the average of three measurements  to determine the number of cells.
?. [Cell preparation]
If using a hemocytometer (count the cells in 1,2,3 and 4 squares in both chambers and take the average). cells/ml = average count per square × 104
?. [Cell preparation]
Total cells = cells/ml × dilution (if used) x total original volume of cell suspension from which sample was taken)
?. [Cell preparation]
Re-suspend the cells in lysis buffer to obtain a concentration of 107 cells/ml
?. [Estimation of tissue homogenate dilution ]
Determine appropriate tissue/cell dilution to be used for the assay.a. Use  pyruvate concentration and plate triplicates of different sample dilutions.b. In a 96-well plate, add  of each TL:LB dilution in triplicate. c. Add  of the pyruvate concentration to each well.
3 µl
147 µl
?. [Cell preparation]
Aspirate the medium from cell culture vessel, and rinse with PBS. Aspirate the PBS, and add 0.25% trypsin in PBS.
?. [Cell preparation]
Place the culture vessel in the incubator (4-10 minutes based on cell type), and after the cells detach, add media (containing serum to inactivate the trypsin).
?. [Cell preparation]
Transfer the cells to a tube and keep some in an eppendorf tube for cell counting.
15 mL
?. [Cell preparation]
Centrifuge tubes at  for  at .
15 mL
Centrifuge: 300 34
4 °C
?. [Pre-preparation]
In a  beaker, measure and add
200 mL
140 mL
?. [Pre-preparation]
Place magnetic stir bar in beaker, place onto stir plate and set to
Centrifuge: 200 33
?. [Pre-preparation]
Measure and record buffer pH (should be , adjust with appropriate base/acid)
?. [Pre-preparation]
In a conical tube, measure and addof dH2O
50 mL
20 mL
?. [Tissue homogenization]
Weigh and record tissue sample ( is sufficient) carefully using sterile forceps (keep frozen). a. Record any additional information about the sample (sample name/description, locus of tissue taken, date, etc.). b. Place tissue into a screw cap vial (compaitble with the bead homogenizer) and place on ice.
6 mg
?. [Estimation of tissue homogenate dilution ]
Prepare varying Tissue/Cell lysate : Lysis Buffer (TL:LB) dilutions (subject to change with varying sample type):a.2X ( TL :  LB)b.5X ( TL : LB) c.10X ( TL :  LB) Keep tissue lysate on ice until ready to pipette into 96 well plate.
100 µl
100 µl
40 µl
160 µl
10 µl
190 µl
?. [Tissue homogenization]
Add  of lysis buffer into the vial with tissue and place on ice
500 µl
?. [Tissue homogenization]
Two options for homogenizing tissueOption 1 - Using bead homogenizer - a. Place a bead (3-5 mm stainless steel) into the bead homogenizer compatible vial (from step 11b) b.  Operate the machine for  at 20–30 HzOption 2 - Using stator-rotor - a. Label plastic tubes EtOH and 0.1% DMPC (dimethyl pyrocarbonate) , one set for each sample) for cleaning.b. Place homogenizer into EtOH for ; wipe excess with Kimwipe.c. Place homogenizer into DMPC H2O for  and wipe excess with Kimwipe.d. Use homogenizer to grind tissue at medium to high settings for approximately  or until tissue is fully homogenized.e. repeat steps b and c before moving to the next sample to clean the homogenizer
?. [Tissue homogenization]
Centrifuge homogenate at for  at
4 °C
Centrifuge: 7000 33
?. [Tissue homogenization]
Transfer supernatant and place into a separate eppendorf tube  labeled with sample name.
?. [Set-up]
Set-up the bead homogenizer in the cold room
?. [Set-up]
Set -up buckets of ice and dry-ice as needed
?. [Set-up]
TECAN spectrophotometer on and set temperature at .a. Ensure the sample plate map of the appropriate method is set for the assay.b. Set-up the protocol for a dynamic  readout at 420 nm and for the appropriate 96 well plate
30 °C
?. [Set-up]
Prepare 12 pyruvate concentrations using pyruvate and reaction buffer by carefully pipeting into pre-labeled conical tubes
?. [Set-up]
Example of dilutions (can be subject to change based on sample type and amount of buffer needed):
?. [Set-up]
Calculate the amount of pyruvate + reaction buffer dilution needed for the entire assay -  x 3 (triplicate) x (# samples)
147 µl
?. [Set-up]
Pipette calculated amount + (to ensure enough is available) into each corresponding 12 well multichannel reservoir-  from lowest to highest pyruvate concentration.
150 µl
?. [Data Analysis]
Do linear regression for each well (change in absorbance over the 10 minutes) to obtain the slope OD/min. (Note: the TECAN Infinity200M accounts automatically for the  path length. So the measurement is actually OD/min/cm)
?. [Estimation of tissue homogenate dilution ]
Place the 96-well plate in the spectrophotometer and measure immediately.
?. [Estimation of tissue homogenate dilution ]
Once the measurement has completed in , Calculate a linear regression for each well to obtain the kinetic output slope (OD/min) and determine the correct dilution by identifying the dilution with the most linear slope.
147 µl
?. [LDH activity measurement]
Pipette  of appropriate TL:LB dilution into three rows (technical replicates) and 12 columns (corresponding to the 12 pyruvate dilutions) on the 96 micro well plate.Note: Ensure that there are no bubbles
3 µl
?. [LDH activity measurement]
Using a  multichannel pipettor, carefully pipette of each 12 pyruvate dilutions in triplicate; assay immediately.Note: Ensure that there are no bubbles
200 µl
147 µl
?. [LDH activity measurement]
Place the 96-well plate in the spectrophotometer and measure immediately.
?. [LDH activity measurement]
Once the read has completed in , click Export to excel and save the file. Eject the plate and clean up
?. [Set-up]
Keep the multi-well reservoir in the  water bath
30 °C
?. [Data Analysis]
LDH enzyme ctivity is calculated as uM NADH/min = OD/min (slope) * Solution Volume in each well () / ( volume of sample lysate () * e )where e=extinction coeff=0.00622 uM-1cm-1Note: take the absolute value of OD/min (omit the negative sign)
150 µl
3 µl
?. [Data Analysis]
Take the average (of the three technical replicates) activity per pyruvate concentration subject to a threshold of Rsq > 0.9
?. [Data Analysis]
Plot Tissue Activity Michaelis-Menten curvea.	X = Pyruvate Concentration (mM)b.	Y = Average Activity
?. [Data Analysis]
For simpler linear curve
?. [Data Analysis]
plot Lineweaver-Burke:a.	X = 1/Pyruvate Concentrationb.	Y = 1/Average Activity
?. [Data Analysis]
Obtain trend-line linear fit in the Excel program and calculate a.	KM = slope / intercept ; [mM]b.	Vmax = 1/ intercept ; uM NADH/minUse Bradford assay kit (https://www.thermofisher.com/order/catalog/product/23200#/23200) to determine protein concentration to express Vmax (LDH activity) in umols NADH/min/mg of protein