Preparation of Embryoid Bodies
Starting materials: Collagenase IV( stock at 10 mg/mL in DMEM)
Non-adherent tissue culture dishes
- Starting with confluent 6 well dish (3.5cm well diameter) of hES cells, aspirate growth media, and rinse with PBS (without Ca++/Mg++).
- Incubate cells for 15 - 30 minutes at 37° C in 2 mg/ml Collagenase IV (in DMEM) to digest extracellular matrix and loosen the hES colonies.
- Once colonies appear to be rounding up at edges, add 2-3 mL DMEM dropwise to center of wells, and rinse gently to dislodge colonies.
- Transfer cells to 15 mL conical tube. Pellet clusters by centrifugation 3 minutes at 500g and gently resuspend in hES growth medium.
- Transfer to non-adherent tissue culture vessel.
Troubleshooting
What to do if after prolonged collagenase treatment hES colonies are not rounding off of tissue culture dish?
To increase the effeciency of the collagenase digestion, the enzyme concentration can be increased to 2-3 mg/mL. Alternatively, although enzymatic treatment is the mildest method of removing ES colonies, mechanical disruption can likewise be used. If colonies are not lifted off following DMEM rinse in the above protocol, colonies can be dislodged by scraping a p1000 tip throughout dish surface or a rubber tipped cell scrappers can be used to remove colonies.
Alternatively, we have found that EB cultures can be established using a very brief trypsonization. Similar to above protocol, rinse cells with PBS, then add 0.5mL 0.05% Trypsin-EDTA/well and incubate approx 1 min. Using a p1000, titurate in dish to dislodge colonies, and transfer to DMEM+10% FBS to stop trypsin. Rinse well with DMEM+10%FBS, and pellet as above.
How to feed suspension cultures of EBs?
During maintenance of EB cultures, the media generally has to be replaced every 2-3 days. This is best accomplished by gently pipetting EBs in their culture media into 15 mL conical tubes, and rinsing wells once to retrieve all EBs. Pellet the EBs either by gravity (let settle for 2-5 min) or centrifugation (3min at 500g). Resuspend EBs in fresh culture media, and return to tissue culture vessel.
Immunostaining of Embryoid Bodies
Starting materials: 10% EM grade formaldehyde (Polysciences #04018)
PBST (PBS + 0.1% Triton X-100)
Blocking solution (10% fetal calf serum in PBST)
Coverslips and adhesive spacers (Secure Seal, Molecular Probes)
- Grow EBs in suspension culture as previously described. Transfer EBs to 4 well dish (1.5cm diameter) for subsequent steps. Manipulation of EBs is best achieved using a p200 pipette.
- Wash EBs briefly with PBS (without Ca++/Mg++).
- Fix EBs for 10 minutes at room temperature in PBS + 4% EM grade formaldehyde.
- Wash twice briefly in PBST.
- Permeabilize in PBS + 0.5% Triton X-100 for 15-30 minutes at room temperature.
- Wash twice briefly in PBST.
- Block for 1 hour at room temperature in blocking solution.
- Dilute primary antibodies according to manufacturer's instructions in blocking solution (minimum volume of 200 uL). Seal dish tightly with lab film, and incubate overnight at 4° C with gentle rocking.
- Wash EBs twice in 500ul PBST, then incubate for 30 minutes at room temperature in PBST.
- Dilute fluorochrome-labelled secondary antibodies in blocking solution (minimum volume of 200 uL), and incubate 1 hour at room temperature in dark.
- Wash twice briefly in PBST.
- Dilute nuclear stain 1/400 in PBST (YOYO; DRAQ5 ; DAPI) + 400 ug/ml RNase A and incubate for 20 minutes at room temperature. For best results, proceed directly to mounting in nuclear stain/PBST/RNaseA mixture.
- Mount EBs between 2 coverslips. Prepare coverslips with adhesive spacer of appropriate thickness. Transfer EBs along with volume of approximately 15-20uL, and gently lay down top coverslip. Store samples in dark prior to confocal imaging.
Troubleshooting
Suggestions for working with EBs if too many are being lost during washing.
Manipulating the EBs is most often the most challenging part of immunostaining. 100uM filter caps designed to filter cells for FACS can be used to prevent EB loss. EBs are placed within these cup shaped mesh filters, and the filters are then moved between wells of PBST for washes.