- Supplemented feeder media (DMEM + 10% FBS+ L-glutamine +
- 50 ml conical tubes
- 1 X feeder freezing media
- 1 ml NUNC cryopreservation vials
- Nalgene isopropanol freezing container(s)
(starting with a 150 mm dish of confluent cells)
- Aspirate media, rinse, and aspirate twice with 25 ml PBS.
- Add 5 ml trypsin/EDTA per dish, roll to cover and place @ 37°C
for 5 min.
- Add 5 ml feeder media; swirl to mix; yielding 10ml/dish. With
25 ml pipette, pipette vigorously several times to break up clumps
and rinse bottom of dish to capture loosely adhering cells.
- Transfer cells from 3 dishes to 50 ml conical tubes and
centrifuge @ 3000 rpm for 5 min.
- Aspirate supernatant, re-suspend pellet in 5 ml feeder
- Pool volumes from all conical tubes into a single 50 ml conical
tube, increase volume to 50 ml, seal cap, and invert to mix.
- Take 1 ml of cell suspension, dilute 1:5 and count
cells. To determine # of vials to stock, divide total cell #
by 5.0 x 106.
- Take remaining 49 ml ON ICE to be irradiated @ 1,000 rads for
Certification is required by the Department of Environmental
Health and Safety in order to operate the irradiator.
- After irradiation, label Nunc cryovials appropriately with
feeder type and batch #. The irradiated feeders are @
- Centrifuge conical tube @ 3000 rpm for 5 min, and aspirate
- Add X mls 1x feeder freezing media (X= # of vials to be
stocked) and re-suspend cells thoroughly.
- Aliquot 1 ml into each vial and close cap tightly.
- Place vials in Nalgene isopropanol freezing chamber (or
Styrofoam box) and put in -70°C freezer O/N.
- Move to liquid nitrogen storage and add to liquid nitrogen
Source: ES Cell Core Lab
Revised: 11/24/03 by Jill Lindner