Alginate Gels (1)

Dominik Haudenschild from Anne, March 2007 at TSRI

Make 2% Protanal LF200 Alginate BATCH 5 with 10mM HEPES and 140ug/ml ascorbate as follows: make as 450ml including 10mM HEPES and ascorbate in water, autoclave 30 minutes on liquid cycle (takes 1 hour), then after autoclaving add rest of filter-sterile buffer solution in 50ml. Final concentrations are: 150mM NaCl=4.01g, 25mM HEPES, 6mg phenol red, 4500mg/L Glucose).

  1.  Make gels, worked best with 3600ul Alginate and 300ul each CaCO3/GDL, fewer bubbles, only a little settling, and strong gel.

  1.  Put cells in gels

  1.  Trypsinize 4 T150 flasks of ~75% confluent P1 chondrocytes. 

  1.  Count, for each 1 ml add the following:

  1.  Resuspend cells in 850ul 2% Alginate

  1.  Add 80ul 1.2M CaCO 3 , mix well

  1.  Add 80ul 0.6M GDL (made at least 2 hours ago)

  1.  Aspirate through 18 guage needle in to 3ml syringe

  1.  Gels should begin to solidify within 90 seconds

  1.  Gels should be solid enough to cut in 10 minute

  1.  Cut sections of gel pushed out of syringe after cutting luer-lock end with razor blade

  1.  Put into incubator "dry" to allow further curing of gel about an hour