Begin the TCPP bead filtration by diluting 0.8 milliliters of the TCPP bead suspension with 3.20 milliliters of ice-cold Hanks'Balanced Salt Solution, or HBSS. Vortex the diluted bead suspension at maximum speed for 15 seconds. Then, remove the plunger from a disposable five-milliliter syringe and fit the syringe with a five-micrometer glass fiber tip filter having a 13-millimeter diameter.
Next, add four milliliters of HBSS to the syringe before adding 0.5 milliliters of the vortexed diluted bead suspension. Using the plunger, filter the bead suspension through the syringe filter at approximately two drops per second. Then, wash the beads by drawing five milliliters of fresh HBSS into the syringe through the filter and pushing the HBSS out into the waste container at approximately two drops per second.
To remove the beads from the filter, draw another five milliliters of fresh HBSS into the syringe through the filter. Then, remove the filter carefully from the syringe. After ejecting the bead suspension from the syringe into a 50-milliliter conical centrifuge tube, place the fresh filter back on the syringe and repeat the five-milliliter HBSS wash four times.
Once done, discard the filter and syringe. Centrifuge the filtered bead suspensions at room temperature for 10 minutes at 1, 000g. Aspirate the supernatant from each 50-milliliter tube.
Gently resuspend the beads and 0.5 milliliters of fresh HBSS. Transfer the combined beads with a P1000 micropipette to a new amber-colored glass vial, and store at four degrees Celsius. The filtration effect on a bead suspension comprising approximately 66%particulates is seen here.
After filtration, the particulate content decreased to approximately 25%The adverse effects of filtration are the loss of beads and a slight loss in fluorescence intensity.
