Technical

Fluorescent Labeling Extracel-X™ Hydrogels

For research use only

To label Extracel-X™ hydrogels either blue or red using Alexa Fluor 350 C5-maleimide or Alexa Fluor 488 C5-maleimide use the following protocol. Note that any fluorescent label can be used so long as it is a C5-maleimide that is thiol reactive.

You will need:

Alexa Fluor 350 C5-maleimide

Thiol-reactive Alexa Fluor® 350 C5-maleimide and its conjugates exhibit an intense, blue fluorescence. Alexa Fluor® 350 protein conjugates are optimally excited at ~346 nm and emit at a wavelength slightly shorter than that of AMCA or AMCA-X conjugates (~442 nm versus ~448 nm).

Alexa Fluor® 488 C5-maleimide

Thiol-reactive Alexa Fluor® 488 C5 maleimide are its conjugates exhibit a bright, green fluorescence. Alexa Fluor® 488 dye, which is spectrally similar to that of fluorescein, is pH-independent from pH 4 to 10.)

INSTRUCTIONS FOR USE

These instructions assume that you will make 1.0 mL of Extracel-X™ that will be labeled with Alexa Fluor® 350 C5-maleimide.

  1. Remove Glycosil™, Gelin-S™, and Extralink™ vials from the -20 °C freezer and heat them to 37 °C (~30 minutes).
  2. Remove the DG Water™ from the -20 °C freezer and thaw in a 37 °C water bath (~15 minutes).
  3. Prepare a stock solution of Alexa Fluor® 350 C5-maleimide in DG Water™ by adding 0.025 mg to 10 µL of water. Note: If you are using a different fluor, verify its solubility in water before proceeding. Note: Make up only as much solution as you will use in one day since they are unstable and light sensitive. Note: Read the manufacturer’s guidelines for making the stock solution to determine if additional steps are required to properly prepare the solution. Note: The optimal pH for reaction of maleimides with thiols is ~7.0.
  4. Under aseptic conditions and using a syringe with the exact amount of liquid, add 1.0 mL of DG Water™ to the Glycosil™ vial. Repeat for the Gelin-S™ vial.
  5. Place both vials horizontally on a rocker at 37 °C (for maximum mixing). It will take <30 minutes for the solids to fully dissolve. Solutions will be clear and slightly viscous. Note: Vigorous shaking will speed up dissolving time.
  6. Under aseptic conditions and using a syringe with the exact amount of liquid, add 0.5 mL of DG Water™ to the Extralink™ vial. Invert several times to dissolve.
  7. As soon as possible, but within four hours of making the solutions, mix equal volumes of Glycosil™ and Gelin-S™. To mix, pipette back and forth or invert the vial.
  8. Protect the dye and hydrogel reaction from light.
  9. For 1ml hydrogel, 0.025mg dye will be needed.
  10. Allow reaction to proceed for two hours at room temperature.
  11. If encapsulating cells, add 200 µL of cells to 2.0 mL of Glycosil™ + Gelin-S™. Pipette back and forth to mix.
  12. To form the hydrogel, add Extralink™ to the Glycosil™ + Gelin-S™ mix in a 1:4 volume ratio (0.5 mL Extralink™ to 2.0 mL Glycosil™ + Gelin-S™). The total hydrogel volume with cells is 2.7 mL.
  13. Gelation will occur within ~20 minutes.