The overall goal of this procedure, is to use reverse phase high performance liquid chromotography to quantify purine metabolism in chronic lymphocytic leukemia or CLL cells measured in different culture conditions. This method has been used to determine when and how cells can produce adenosine in the extracellular environment. And this is a critical issue, as adenosine is both a cytoprotective and immunosuppressive molecule.
Furthermore, it requires monumental culture medium and simple preparation is extremely easy. We first had the idea for this method when we started studying purine metabolism in the tumor micro environment. And we realized that it was essential to have an easy and reproducible way to measure this metabolism using primary cells.
Make a one to three dilution of whole blood with room temperature one x phosphate buffered saline or PBS, this step is needed to obtain purified CLL lymphocytes. Then used to measure extracellular purine metabolism. Purified peripheral blood mononuclear cells from blood samples by density gradient centrifugation.
First underlay five milliliters of density centrifugation media in a 15 milliliter centrifuge tube. And carefully transfer 10 milliliters of diluted blood. Immediately centrifuged at 1, 500 times G for 20 minutes at room temperature.
Aspirate part of the supernatant with a glass pasture pipette connected to a vacuum pump. Then collect the PBMC ring at the interface, between the density centrifugation media and the diluted plasma with a five milliliter pipette. And transfer the cells to a 15 milliliter centrifuge tube.
Wash the cells with PBS. Centrifuge at 500 times G for five minutes at four degrees celsius. Discard the supernatant and resuspend 10 million PBMC's in one one milliliter of isolation buffer.
Which is PPS, with a zero point one percent BSA and two millimolar EDTA with a pH adjusted to seven point four. CLL lymphocytes now need to be purified from the PBMC preparations. Then, add 10 micrograms of mouse monoclonal anti-CD3, CD14, and CD16 primary antibodies, and incubate for 30 minutes at four degrees celsius.
After washing the cells with the isolation buffer, and centrifuging at 500 times G for five minutes at four degrees celsius, resuspend the 10 million PBMC's in one milliliter of isolation buffer. Incubate the re-suspended cells with 100 microliters of washed magnetic beads for 30 minutes at four degrees celsius with gentle re-suspending. Then place the tube in the magnet holder for two minutes.
And transfer the supernatant with the unbound cells to a fresh tube with a five milliliter pipette. After this step, purified CLL lymphocytes are ready to be used. To set up the HPLC, first prepare two liters each of buffer A and buffer B as described in the text protocol.
Then, connect the guard column and the column to the HPLC. Log into the HPLC software, and select the Browse Project button. Navigate to the file menu, and select New Method, and then Instrument Method.
Program the equilibration column method as listed in the text protocol. Set a flow rate of 1.00 milliliters per minute and program the ultraviolet detector to read at 250 nanometers. Save the method.
Repeat this procedure to program the run sample method indicated in the text protocol. Set a flow rate of 1.00 milliliters per minute and program the ultraviolet detector to read at 260 nanometers. Save the method.
Select the Run Samples button and choose New Sample Set Method from the File menu. Select the empty option and enter the number of the vial in the auto-sampler, the sample name, and the injection volume of 50 microliters. Select the method set, saved for the method set column, and enter the total run time in minutes.
Enter the equilibration column method in method set column for each sample run. And enter the total run time. Proceed to generate a standard calibration curve for each compound, as described in the text protocol.
To test AMP consumption, and adenosine and inosine generation, re-suspend two million CD19 positive CD5 positive CLL cells in 250 microliters of serum free media. And plate the cells in a 48 well plate. Add 250 microliters of 400 micromolar AMP, or AMP plus inhibitors to obtain a final concentration of 200 micromolar AMP.
Include a condition in the absence of the substrate to be used as a blank sample. Incubate from the 30 to 60 minutes at 37 degrees celsius. At the end of the incubation time, collect 500 microliters of the supernatants in cold microcentrifuge tubes.
Immediately centrifuge at 17, 000 times G at four degrees celsius for five minutes. Transfer the supernatants in a new 1.5 milliliter microcentrifuge tubes, and either proceed to sample preparation for the HPLC runs or immediately store at minus 80 degree celsius. Following pre-treatment with the inhibitors, an incubation with AMP substrate, filter the supernatants in new 1.5 milliliter microcentrifuge tubes with 0.2 micron syringe filters.
Use one milliliter syringes for filtering. If the HPLC system is provided with an auto-sampler, prepare the glass vials for HPLC and use the 100 microliter micro-inserts for small volumes of sample. Transfer at least 100 microliters of sample in the glass vial with a micro-pipette, or a glass pasture pipette.
Be careful to transfer without bubbles and close the vial with the screw cap. Samples are now ready for analysis by reversed phase HPLC. Select the Run Samples button and choose New Sample Set Method from the File menu.
Select the empty option and indicate the number of the vial in the auto-sampler, the sample name, and the injection volume of 50 microliters. Select the equilibration column method and the run sample method described in the text protocol. For all the blank and sample runs that follow.
Then inject 50 microliters of the blank of serum free media followed by each sample. To determine the concentration of purines in each sample, quantify the AMP, adenosine, and inosine by obtaining the peak areas at the retention times described in the text protocol. Select the Process button, followed by the Integrate option.
Choose the start and end points of each single peak to obtain the area measurement. This is done manually by tracing a line between the start and end points of the peak. Proceed to calculate the AMP, adenosine, and inosine consumed and or produced by the chronic lymphocytic leukemia cells, as described in the text protocol.
The percentage of leukemic cells is evaluated by flow cytometry after gaining on live cells stained with anti-CD19 and CD5 antibodies. Before the negative isolation of B cells, the percentage of CLL cells corresponds to the 75%After purification, the percentage of CLL B lymphocytes reaches 95%Shown here, are representative results of adenosine and inosine generated by CD73 positive leukemic lymphocytes after 30 minutes of incubation with AMP as substrate. Pre-treatment with the CD73 inhibitor, APCP, completely blocks extracellular adenosine synthesis.
Here is an HPLC chromotogram obtained from supernatants of CD73 positive CLL cells. Pre-treated or not with the adenosine deaminase inhibitor, ENHA hydrochloride before 30 minutes of incubation with AMP. Results indicate blockade of adenosine conversion to inosine.
The same results are confirmed by pre-treatment of CLL cells with deoxycoformycin, or DCF, another adenosine deaminase inhibitor. Once mastered, this technique can be done in one day if it is performed properly. And it can be immediately stored in and analyzed later.
After watching this video, you should have a good understanding of how to measure excess purine compounds and cell culture media from purified human leukemic cells. The use of enzyme inhibitors serves as internal control. While attempting this procedure, it's important to remember that generation of good standard calibration codes for each compound is a critical issue to have an accurate quantification.
Alternative or complimentary to this, investigators can use the reverse phase HPLC without the maximum detection or faster by more indirect methods such as monoclonal to green phosphate assay or the cypherus base method. After it's development, this technique paved the ways to researches for investigating purine metabolism and active enzyme action in the context of solu-tumors or hematoligcal malignancies and to correlate these characteristics to clinical or molecular features of the disease. Don't forget that working with patient derived material, organic solvents and the hydrochloric acid can be extremely hazardous.
Precautions such as lab coat, gloves, and the use of a chemical hood should always be taken while performing this procedure. The implications of this technique are relevant to cellular biology and potentially to therapy. Because patients that produce adenosine are generally characterized by an inferior prognosis.
This method has been applied to CLL lymphocytes, however in principle it can be applied to any kind of primary cells, it's line, interdepense or in suspension.
