LDH by stariya

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									                                                                   Raymond A. Swanson, M.D. 4/26/91
                                                                                 revised 1/7/92
                                                                                 revised 2/5/94
                                                                                        revised 12/26/00

                      LDH CELL DEATH ASSAY ON PLATE READER

    NADH + pyruvate                   NAD + lactate
                           LDH

The assay measures NADH absorbance at 340 nm. Under saturating conditions, rate of absorbance decline
is proportional to LDH activity. Rate will become non-linear after a relatively short period (2 to 5 minutes)
because substrates are consumed and products accumulate.

Materials:
      96-well plate
      Multiwell pipettor
      Molecular Devices plate reader (with Softmax software)

Stock solutions
       1) Assay buffer - 500 mM potassium phosphate buffer pH 7.5
       2) 100 mM pyruvate, frozen in 1ml aliquots in assay phosphate buffer
       3) NADH, 5mg / 20ml buffer (enough for 200 wells), made fresh on day of assay
       4) Lysing buffer - 3% Triton X, 5 mM phosphate buffer pH 7.5
         - Can be prepared as 10x stock with 30% Triton X, 50 mM potassium phosphate buffer pH 7.5

Samples
      Monotype or mixed cell cultures


Procedure A: Mixed cultures.
    Assay of LDH in supernatant can be used for cultures of multiple cell types (e.g. neurons and
    glia) if a) it is known that only one cell type (e.g. neurons) is dying, b) it can be assumed that
    equal numbers of neurons were plated in each well at beginning of experiment, c) there is a way
    to produce 100% neuronal death without killing the other cell types, and d) sufficient time has
    elapsed to allow LDH efflux from dead (ruptured) cells. This is about 18 hours for excitotoxic
    neuronal death, but as short as 20 minutes for direct oxidative injury.
1) Sample groups -
      a) Controls (generally wash only)
      b) Experimental
      c) 100% neuronal death
2) Allow all solutions to warm to room temperature
3) Add 0.5ml of 100mM pyruvate stock to the phosphate buffer / NADH solution
4) Collect 100 l supernatant from each culture well and place in a 96-well plate. Use triplicates if
      possible.
5) Place 100l of pyruvate/NADH/buffer solution to each well using a multiwell pipettor
6) Immediately read plate using the “LDH-neg” program (see below**)

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Data analysis
1) Check for linear plots in each well. Discard wells in which r2 is less than 0.85.
2) Download Vmax data to an excel spreadsheet
3) Calculate the means of all triplicate determinations.
4) Calculate the mean Vmax of the control (wash only) wells. This is value is Xc
5) Calculate the mean Vmax of the 100% neuronal death wells. This value is Xd
6) For every well (including the control and 100% death wells), transform the Vmax value to % neuronal
      death by this formula: % dead = 100 x (Vmax - Xc) / (Xd - Xc)
7) Use these normalized values to calculate means and s.e. for each experimental and control group.


Procedure B. Monotype cultures from cells that easily lift off
    Assay of LDH can be used for monotype cultures even if differing cell numbers were present at
    the beginning of the studies and even if some live or dead cells have become detached.
1) Sample groups -
      a) Controls (generally wash only)
      b) Experimental
2) Allow all solutions to warm to room temperature
3) Add 0.5ml of 100mM pyruvate stock to the phosphate buffer / NADH solution
4) Sample preparation:
  a. Label one set of centrifuge tubes for each culture well.
  b. Collect all the medium from each well and place in the centrifuge tubes
  c. Spin to precipitate any floating live cells
 d. To the culture wells, add lysing buffer (volume equal to that of the media aspirated from each well).
      Let stand until cells are lysed (5 to 30 minutes, faster if placed at 37º)
 e) From the spun centrifuge tubes, place 100l medium in each of three wells in a 96-well plate. (This
      contains the extracellular LDH, from the cells that were dead (ruptured) at the time of sample
      collection.) Discard the rest of the medium, but do not discard the centrifuge tubes.
 f) After cells in the plates are lysed, aspirate all of the lysing buffer from each well and place in the
      corresponding centrifuge tube that contains the cell pellet from the supernatant.
 g) Triturate to re-suspend and lyse any live, unruptured cells in cells the pellet, then Vortex gently.
 h) Place 100l of the lysed cells into each of three wells in a 96 well plate. (This contains the intracellular
      LDH, from the cells which were alive (unruptured) at the time of sample collection).
5) Place 100l of pyruvate/NADH/buffer solution to each well using a multiwell pipettor
6) Immediately read plate using the “LDH-neg” program (see below)**
Data analysis
1) Check for linear plots in each well. Discard wells in which r2 is less than 0.85.
2) Download Vmax data to an Excel spreadsheet
3) Calculate the means for all triplicates determinations.
4) For each well, calculate the % cell death by this formula: (100 x Vmax extracellular) / Vmax
      intracellular.
5) Use these normalized values to calculate means and s.e. for each experimental and control group.




Procedure C: Monotype cultures from cells that are firmly adherent (e.g. astrocytes)
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    For this cell type it can be assumed that no living cells are floating in the medium. This
    simplifies the procedure since it is then not necessary to centrifuge the medium to remove live,
    floating cells.
1) Sample groups -
      a) Controls (generally wash only)
      b) Experimental
2) Allow all solutions to warm to room temperature
3) Add 0.5ml of 100mM pyruvate stock to the phosphate buffer / NADH solution
4) Sample preparation:
  a. Place 100 l of medium from each well into each of 3 wells in a 96-well plate. This is contains the
      extracellular LDH, from cells that are dead (ruptured) at the time of sample collection.) Discard the
      rest of the medium.
  b. To the culture wells, add lysing buffer (volume equal to that of the media aspirated from each well).
      Let stand until cells are lysed; 5 to 30 minutes, faster if placed at 37º. (This contains the intracellular
      LDH, from cells that were alive (unruptured) at the time of sample collection.)
  c. Triturate once or twice to be certain cells are broken, then transfer 100 l of the lysates from each well
      into each of 3 wells in a 96-well plate.
5) Place 100l of pyruvate/NADH/buffer solution to each well using a multiwell pipettor
6) Immediately read plate using the “LDH-neg” program (see below)**
Data analysis
1) Check for linear plots in each well. Discard wells in which r2 is less than 0.85.
2) Download Vmax data to an Excel spreadsheet
3) Calculate the means for all triplicates determinations.
4) For each well, calculate the % cell death by this formula: (100 x Vmax extracellular) / Vmax
      intracellular.
7) Use these normalized values to calculate means and s.e. for each experimental and control group.



**LDH-neg Protocol
Key points are the following:
a) Wavelength = 340 nm
b) Kinetic read, time = 75 seconds
c) Automix 5 seconds before first read.
d) No pre-read of plate
e) Minimum O.D. = -0.06, Maximum O.D. = 0.0
f) Display raw O.D., show with reduced number of points.




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