Isolating peripheral blood mononuclear cells, also known as PBMCs, is typically a lengthy and manual process. We saw an alternative method that'd be quick to implement and capable of processing high numbers of samples. Our protocol offers an automated high throughput method, ensuring consistency while preventing technician burnout.
We also detail our medium throughput automation compatible method that can be utilized during equipment downtime. This medium throughput option is a more budget-friendly alternative for other laboratories. A biobank can now process more samples with the same number of technicians.
This will increase the specimen collection size as well as its diversity, enabling future research and improving patient care. To begin, gently invert 10 milliliter whole blood tubes 10 times at room temperature. Using a swinging bucket rotor, centrifuge the tubes at 800 G for 10 minutes at 22 degrees Celsius with the break on.
Then place the samples in a biological safety cabinet and check for the three distinct layers. Label three five-milliliter polystyrene tubes with letters A, B, and C.Swirl and collect one milliliter of the buffy coat by aspiration and transfer to the tube labeled as A.Then add 60 microliters of 0.1 molar EDTA to tube A containing the buffy coat. Add 50 microliters of the cocktail mix to tube A.Pipette up and down at least three times to mix it and incubate for five minutes at room temperature.
After that, add 890 microliters of PBS to tube A and mix by pipetting at least three times. Next, vortex the magnetic bead tube for 30 seconds. Add 50 microliters of the magnetic beads to tube A and pipette at least three times to mix the beads thoroughly.
Immediately place tube A into the magnet stand and incubate for five minutes at room temperature. Then carefully pipette the enriched cell suspension into tube B, collecting the clear fraction with no or minimal red blood cells for optimal PBMC recovery. After that, remove tube A from the magnet stand and dispose of it.
Next, add 50 microliters of magnetic beads to cell suspension in tube B and pipette up and down at least three times. Immediately place tube B into the magnet stand and incubate for five minutes at room temperature. Carefully pipette the enriched cell suspension into tube C, collecting only the clear fraction.
Remove tube B from the magnet stand and dispose of it. Then immediately place tube C into the magnet stand and incubate for five minutes at room temperature. Carefully pipette the enriched cell suspension into a labeled centrifuge tube and top up to two milliliters with PBS.
Transfer 50 microliters of the cell suspension to a sample cup of the automated cell counter. Add 450 microliters of PBS for a 1-to-10 dilution cell count. To begin, label two 14 milliliter tubes as A and B, one 15 milliliter centrifuge tube as C, and one 15 milliliter centrifuge tube as waste.
After centrifuging the whole blood, collect and add one milliliter of the prepared buffy coat to tube A.Add 60 microliters of 0.1 molar EDTA to tube A.Then vortex the magnetic bead tube for 30 seconds. To turn on the automated PBMC instrument, switch the power on at the front of the instrument. On the instrument's home screen, select Profile and choose the desired protocol.
Then enter the starting volume and repeat for each sample. Select all the quadrants that will use the same reagent kit. Next, load labeled consumables, filter tips, and buffer container into each quadrant of the instruments carousel as directed on the automated PBMC instrument screen.
Once loading is complete, remove lids from consumables and reagents and select run on the instrument screen. When the run is complete, select unload and remove samples from the instruments carousel. Transfer 500 microliters of the cells to a sample cup for a no dilution cell count.
Comparing the automated bead-based method to the manual method, no significant differences were identified in cell viability or total cell counts. The manual method processed eight samples in 43 minutes, while the automated method took 57 minutes, including 35 minutes of hands-off processing. Mean cell viability was greater than 90%for PMBCs processed within five days, and greater than 75%for those processed within 10 days.
Mean PBMC yields decreased by 50%after five days compared to specimens processed within 24 hours.
