Refolding Protocol for CD133-A15, 5'-DY647

This is intended as a guide only; for full experimental details please read the reference provided.

Binding Data

CD133-A15, 5'-DY647 (Cat.No. 6103) has Kd values of 33.9 and 83.2 nM at CD133, in Hep3B cells and HT-29 cells respectively, as measured by flow cytometry (see figure).

Binding Data

Reconstitution and Refolding Protocol

Reconstitute the aptamer at 100 µM in sterile dH2O and ensure the aptamer has fully dissolved. We recommend that stock solutions, once prepared, are stored aliquoted in tightly sealed vials at -20°C or below and used within 1 month. Wherever possible solutions should be made up and used on the same day. Repeated freeze-thaw cycles should be avoided.

Prior to use, dilute the aptamer at an assay-dependent concentration in PBS buffer containing 2.5 mM MgCl2. Heat the solution at 85˚C for 5 min, incubate for 10 min at room temperature and finally allow to refold for 15 min at 37˚C.

Unfixed Cell Imaging Protocol

(This protocol is also applicable to flow cytometry)

Twenty-four hours prior to labeling, seed cells at a density of 75,000 cells per cm2 in an 8-chamber slide (Lab-Tek II, Nunc). Remove media and incubate cells in blocking buffer (PBS buffer containing 2.5 mM MgCl2, 0.1 mg/mL tRNA (R5636, Sigma), and 5% FBS) at 37 ˚C for 15 min. Wash twice in binding buffer (DPBS with 2.5 mM MgCl2, 0.1 mg/mL tRNA and 0.1 mg/mL salmon sperm DNA) prior to incubation with 200 nM (1/500 dilution) refolded aptamer (typically 100 µL) for 30 min at 37 ˚C. Additional step for unfixed cell imaging only: add Hoechst 33342 (3 µg/mL) to the cells during the final 15 min of incubation. Remove the aptamer solution and wash three times for 5 min each in binding buffer prior to visualization.

Reagent Tocris Cat. No.
PBS 5564
Hoechst 33343 5117

Imaging in Unfixed Tumorspheres Protocol

Wash spheres three times in PBS containing 2.5 mM MgCl2 and block for 20 min using blocking buffer (PBS buffer containing 2.5 mM MgCl2, 0.1 mg/mL tRNA and 5% FBS). Incubate spheres with 100 nM (1/1000 dilution) refolded aptamer for a minimum of 30 min. Wash spheres three times with PBS prior to visualization.

This protocol has been produced in collaboration with Dr Sarah Shigdar, Deakin University, Australia.

Reference

Shigdar et al. (2013) RNA aptamers targeting cancer stem cell marker CD133. Cancer Lett. 300 84. PMID: 23196060

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