Shreffler: Fluorochrome Beads

Materials

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.

References

1. Savina A, Vargas P, Guermonprez P, Lennon AM, and Amigorena S. Measuring pH, ROS production, maturation, and degradation in dendritic cell phagosomes using cytofluorometry-based assays. Methods Mol Biol. 2010;595:383-402. DOI:10.1007/978-1-60761-421-0_25 |