PBMC and plasma samples are straightforward to acquire and highly informative in early clinical development. This robust protocol minimizes the pre-analytical variability introduced at clinical sites. This protocol is fast and easy, requiring basic lab equipment and minimal operator expertise to process the blood into PBMCs and plasma.
It is compatible with busy lab days at hospitals. Downstream analysis of blood components resulting from this protocol can inform pharmacokinetic and pharmacodynamic relationships, drug dose scheduling and proof of target engagement and provide insights into drug mechanisms of action, supporting both real-time and introspective analysis. We use this protocol for oncology studies, specifically to analyze DNA damage response mechanisms.
However, materials collected by this protocol can be relevant across many disease areas and multiple therapeutic modalities. After receiving eight milliliters of donor blood per sample, record the time at which the blood was drawn and gently invert each tube eight to 10 times before transferring the anticoagulant treated blood samples into each of the tubes labeled for radiation. For clinical samples, centrifuge the tubes right after inverting them.
Place the 0.2 gray tubes in a pre-warmed x-ray cabinet, close the door and apply a 0.2 gray dose to the tubes. Replace the low-dose irradiated tubes with the seven gray tubes and adjust the dose to apply a seven gray radiation dose to the high-dose irradiation samples. After both samples have been irradiated, incubate all three of the tubes at 37 degrees Celsius for one hour.
Transfer the samples to cell preparation tubes before separating the cells with high-speed centrifugation, making sure to set the units as x g or equivalent RCF. After the centrifugation, view the tubes against a dark background to identify the plasma, red blood cell, and buffy coat layers. Using a serological pipette, transfer approximately half of the plasma into a labeled 15 milliliter conical centrifuge without disturbing the buffy coat layer and store the tube of plasma on wet ice.
Then, use a pasture pipette to transfer the entire PBMC containing buffy coat layer into a new 15 milliliter tube and add room temperature PBS to the PBMC to a final volume of 15 milliliters per sample. Gently mix the cells by inversion five times and collect the cells by centrifugation at a lower speed, making sure to check the centrifugation units. Aspirate all but the last 500 microliters of supernatant without disturbing the pellets.
Add fresh PBS to a final volume of 10 milliliters to each tube and resuspend by gently inverting the tubes. Use 40 microliter aliquots of the cells for counting. After counting, collect the cells with another centrifugation and remove as much supernatant as possible without disturbing the pellets.
To store the PBMC for later processing, resuspend the pellets in 50 microliters of fresh PBS per sample with gentle pipetting and transfer each entire cell suspension into a labeled 1.5 milliliter cryovial on wet ice. Then flash freeze the cells by the preferred method and place them in a freezing box at minus 80 degrees. Record the time at which the cells were frozen.
Alternatively, resuspend the pellets in one milliliter or freezing mixture per sample and transfer the cell suspensions to individual labeled cryovials. Then placed the tubes and pre-chilled freezing boxes for minus 80 degrees Celsius storage. To freeze the plasma, add up to one milliliter aliquots lots of the plasma to up to each of five two milliliter microtubes without disturbing the residual pellet and record the total volume of plasma in each tube.
Then, immediately freeze the plasma aliquots in an upright orientation in a minus 80 degrees Celsius freezer. For Western blot analysis of the samples, resuspend the pellet in each tube with an appropriate volume of lysis buffer supplemented with protease and phosphatase inhibitors equal to that of the cell pellet volume with gentle pipetting and incubate the cell samples for 10 minutes on ice. At the end of the incubation, transfer the samples to 1.5 milliliter tubes and sonicate them with three 30 seconds on 30 seconds off cycles at four degrees Celsius.
Collect the samples by centrifugation and transfer the supernatants to new 1.5 milliliter tubes. Then, mix 40 micrograms per sample with loading buffer and boil the samples for five minutes. Load 20 micrograms of each sample per lane in duplicate in a 4-12%Bis-Tris protein gel and run an SDS-PAGE.
The number of cells obtained from eight milliliters of blood varies according to the patient and disease setting. In general, the cell pellet is small in size and transparent white in color. Sometimes the pellets can have some red blood cell contamination, which has a negative effect on the quality of the preparation.
A typical yield from a chronic lymphocytic leukemia patient using this protocol varies from 1.62 times 10 to the fourth to 1.99 times 10 to the ninth cells per eight milliliter mononuclear cell preparation with a final protein concentration ranging between 1.62 to 19.77 milligrams per milliliter. In this representative analysis of three patients samples, an increase in post-translational modifications was observed at higher ionizing radiation doses. Interestingly, the phosphorylation of ataxia-telangiectasia mutated and RAD50 was substantial at the low dose of 0.2 grays, which suggests these post-translational modifications may be feasibly interrogated as pharmacodynamic biomarkers for treatments involving the generation of DNA double strand breaks with a good dynamic range in tumor, as well as peripheral blood samples.
For an optimal sample preparation, process the blood within one hour of collection at room temperature, centrifuge the samples correctly, and view the tube against the dark background to be able to distinguish the buffy coat.
