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 instrument's 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 instrument's 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 PBMCs 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.
