The overall goal of the following experiment is to determine intracellular or extracellular ascorbate levels in cultured or isolated cells. This is achieved by incubating cell extracts or whole cells with a score oxidase to selectively deplete a score BA in subsequent steps. In the first method, cell extracts are incubated with cyanide to generate ferro cyanide in an ascorbate dependent manner, which gives a direct representation of the amount of ascorbate in the extracts.
Next, the amount of ferro cyanide formed in the cell extracts is determined by quantitatively oxidizing the ferro cyanide with added ferric iron to form Ferris iron in order to generate a chromogenic iron two ferne S complex that can be quantified spectro photometrically. In the second method, all cells are directly incubated with the iron two chelator ferne S and ferric citrate. The results show differences in intracellular or extracellular ascorbate levels based on the quantitative and specific reduction of extracellular iron complexes that can be detected.
Spectro metrically. The main advantage of the current technique over methods that are already in place like HBLC based detection is that this highly specific microplate assay can be carried out very quickly and all samples and standards can be processed in parallel rather than in a serial manner Prior to starting this procedure. Culture K 5 62 suspension cells and create in a score BA containing cellular extract.
Following this, prepare a stock solution of 10 millimolar ascorbic acid in ice cold PBS. Then carefully prepare a series of ascorbate standards between zero and 20 micromolar. Carefully remove four 100 microliter aliquots from each standard and add them to a horizontal sequence of wells.
In a 96 well flat bottom plate that contains 25 microliters per well of PBS or a 45.5 units per milliliter. Stock solution of L ascorbate oxidase or AO in PBS cover the 96 well plate with foil and orbital. Mix it at 550 RPM at room temperature for five minutes.
When finished, add 50 microliters of 3.5 millimolar potassium Ferris cyanide in PBS to all wells to give a final Ferris cyanide concentration of one millimolar after orbital mixing the 96 well plate, using the same conditions as before. Immediately add 25 microliters of a freshly constructed solution containing 50%acetic acid and 30%trichloroacetic acid. Next, add 100 microliters of a freshly prepared Pharaoh cyanide determination solution to each well orbital.
Mix the plate in the dark at 550 RPM for 30 minutes at room temperature. Then read the absorbance values of the wells at 593 nanometers. To begin the assay.
First oxidize extracellular ascorbate in selected wells by adding 50 microliters of 120 units per milliliter AO or HBSD to paired wells in triplicate to give a final AO concentration of 10 units per milliliter. Following this label, the paired wells as minus AO and plus ao. After mixing the plates with gentle orbital mixing for five minutes at 37 degrees Celsius, add 50 microliters of 2.4 millimolar Ferne S to all of the wells to give a final ferne s concentration of 200 micromolar, mix the plates as above for five minutes at 37 degrees Celsius.
Next, add 50 microliters of freshly prepared 120 micromolar ferric citrate to all of the wells to give final iron and citrate concentrations of 10 micromolar iron and 50 micromolar citrate respectively. Mix well at the end of the ascorbate FLX assay. Rapidly aspirate the overlying medium from each well and add to appropriately labeled wells in a 24 well plate.
Following this, add three microliter aliquots of the supernatant to a 96 well plate to determine extracellular ascorbate. Read the absorbance values of the wells at 593 nanometers in a microplate spectrophotometer. Finally, calculate the amount of extracellular ascorbate as m ascorbate per milligram protein shown.
Here is a typical standard curve for a set of ascorbate standards, zero to 20 micromolar or zero to two NMO ascorbate per well. Of the 96 well plate, although not shown here, linearity is maintained up to eight NMO ascorbate per well, which corresponds to a sample ascorbate concentration of about 80 micromolar. The assay can be used to successfully detect ascorbate levels below 0.25 ole ascorbate per well.
K 5 62 cells rapidly accumulate intracellular ascorbate from extracellular dehydro L ascorbic acid, but not ascorbate. In this representative experiment, PBS washed K 5 62 cells were incubated in PBS with 500 micromolar of either ascorbate dehydro L ascorbic acid dehydro L ascorbic acid plus 50 micromolar cyto B ascorbate plus five millimolar cyanide or ascorbate plus 50 units per milliliter AO for 30 minutes at 37 degrees Celsius. Intracellular ascorbate was determined as described previously.
Dehydro L ascorbic acid uptake by K 5 62 cells occurs by facilitative glucose transporter glute mediated transport to assess the involvement of glutes in dehydro L ascorbic acid uptake. In this representative experiment, K 5 62 cells were incubated with increasing concentrations of cyto B or dihydro cyto B prior to incubation with dehydro L ascorbic acid. It is important to note, while both cyto callin B and dihydro cyto B inhibit motile processes at micromolar concentrations only cyto callin B, which differs from dihydro cyto B by the presence of a single double bond inhibits glute dependent transport at low micromolar concentrations.
Therefore, as dehydro L ascorbic acid uptake was inhibit by cyto B with an IC 50 of less than 2.5 micromolar, but was not inhibit by dihydro cyto. B dehydro. L ascorbic acid uptake probably occurs by glute mediated transport.
Alternatively, washed cells were exposed to increasing concentrations of the glute transportable, but non metabolizable glucose analog three O methyl D glucose or the non glute transportable glucose stereo isomer L glucose during incubation with dehydro L ascorbic acid. Again, the results indicate glu involvement in dehydro L ascorbic acid import as inhibition of intracellular ascorbate accumulation occurs only in the presence of the glute transportable glucose analog. In the second assay.
The rate of ascorbate efflux from cultured cells can be determined. This specific assay is important as the release of ascorbate from cells appears to be crucial for the ascorbate regulated cellular uptake of non transferrin bound iron by cells. The uptake of non transferrin bound iron is considered to be relevant to the pathophysiology of iron overload disorders, such as the hemochromatosis, as well as to astrocyte neuron iron exchange and homeostasis in the mammalian brain.
Indeed, the major excitatory neurotransmitter L glutamate triggers the release of ascorbate for astrocytes in a manner that depends on L glutamate, uptake by glass, and probably GLT one and subsequent cellular swelling that triggers the release of ascorbate by putative VSOs. In analogy, A score bate release from A score B loaded astrocytes can be stimulated by the excitatory amino acid L aspartate, but not the non excitatory amino acid L glutamine. This effect is depicted here, which shows dose response curves for the stimulation of ascorbate release by aspartate and glutamine from primary cultures of ascorbate loaded mouse astrocytes Once mastered.
This technique can be performed in as little as two hours if performed correctly.
