Shreffler: Fluorochrome Beads

Equipment

 * Microcentrifuge
 * Rotator

Reagents

 * Sodium hydrogen carbonate 0.1 M buffered at pH 8.5 with NaOH (can mix 0.3360 g NAHCO3 w/ 40 ml DIH20)
 * Glycine 0.1 M in PBS (can mix 0.3754 g glycine w/ 50 ml PBS)
 * FITC (F7250-100MG; Sigma-Aldrich Inc), aliquotted at 50 mg/ml, 50 µl/aliquot)
 * Polybeads Amino 3.0 Micron Microspheres, 5 mL (Cat# 17145; Polysciences, Inc)

Procedure

 * 1) Place 50 µl Polysciences Polybeads into eppendorf tube.
 * 2) Add 0.5 ml sterlier PBS and cap tightly.
 * 3) Centrifuge for 2 min in microcentrifuge at 15,000 rpm. Aspirate supernatant using Pasteur pipette.
 * 4) Resuspend pellet in 400 µl of sodium hydrogen carbonate 0.1 M buffered at pH 8.5. Vortex until pellet is dispersed.
 * 5) Add 50 µl of 50 mg/ml FITC solution (from aliquots) to the bead solution. Cap tightly and cover tube with aluminum to protect from light.
 * 6) Leave on rotator for 2 h at room temperature.
 * 7) Centrifuge for 2 min in microcentrifuge at 15,000 rpm. Aspirate supernatant.
 * 8) Resuspend pellet in 0.5 ml of 0.1 M glycine in PBS. Vortex until pellet is dispersed. Add an additional 0.5 ml of PBS 0.1 M glycine.
 * 9) Centrifuge for 2 min in microcentrifuge at 15,000 rpm. Aspirate supernatant.
 * 10) Repeat 8 and 9.
 * 11) Resuspend pellet in 0.5 ml PBS. Vortex until pellet is dispersed. Add 0.5 ml PBS to obtain a total volume of 1 ml.
 * 12) Centrifuge for 2 min in microcentrifuge at 15,000 rpm. Aspirate supernatant.
 * 13) Rsesuspend pellet in 40 µl of sterile PBS to obtain a bead dilution equivalent to the initial (1.7 x 10^6 beads per µl for 3 µm amino beads).
 * 14) Cap tightly and cover tube with aluminum foil to protect from light. Beads are ready to use and can be stored at 4°C for a few days. Do not freeze.