Shreffler: Fluorochrome Beads

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Contents

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. . pmid:19941126. PubMed HubMed [Savina]
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