<p>Hanging Drop Method</p><p>DAY 0 Take a solution of ES cells diluted to 25,000cells/ml (Differentiation medium here) – in a 50ml falcon Calculate the number of plates you need for this step Label that number of bacterial plates appropriately and fill the tray (bottom) part with PBS</p><p>Take a P100/200 and set it to 25µl</p><p>The Hanging drops are placed on the lids of bacterial dishes. Before starting stir well the 50ml falcon tube that your cell dilution is in</p><p>Place 5 to 10 drops on the lid before shaking again, continue this process until you have placed 40-50 drops onto the plate lid. </p><p>Once this has been accomplished, turn over the lid in a smooth and continuous motion placing it on its tray counter part</p><p>After making three hanging drop plates place them in the incubator Leave them there for two days (FOR PROPER HANGING DROP FORMATION, THE DROPS MUST NOT BE DISTURBED FOR A TWO DAY DURATION)</p><p>Image below shows what a hanging drop plate should look like</p><p>Hanging drops with 4-500 ES cells per drop</p><p>5 ml of PBS in the bottom part of the bacterial dish</p><p>DAY 1 -- Nothing here</p><p>DAY 2 – Resuspention of ES cells</p><p>Keeping in mind the number of bacterial plates you made in the hanging drop step, place 8 ml of Differentiation medium in the same number of plates and label them appropriately</p><p>Place 2 ml of differentiation medium into a 5ml pipette and resuspend all of the drops on the lid. This must be done with very gentle flushes so as to not disturb the delicate architecture of the Embryoid body (EB). Also, bubbles generated by pipetting should be avoided.</p><p>Placed resuspended EB’s into the bacterial dishes with 8 ml of medium</p><p>Leave them once again for 3 days</p><p>DAY 5 – Plating ES Cells</p><p>Plate about 20 EB’s in a 100mm dish (if using smaller dishes use 10 EB’s in a 65mm dish, 5 EB’s in a 35 mm dish). With this estimate the number of dishes you will need, gelatin coat them with 0.1% gelatin, leave for at least one hour. Dry the gelatin coated plates (vacuum the gelatin off, leave in hood with lid off) Place Differentiation medium in the plates (10ml for 100mm, 5ml for 65mm, and 2ml for 35mm) VERY GENTLY, pick up EB’s with 1ml pipette (place the pipetter on setting 2) from the bacterial dish, and place them on to the gelatin coated dish, one can pick us several EB’s at a time…</p><p>DAY 6 – Plated ES</p><p>Some ES cells will not stick to the Gelatin coated plates, therefore, the medium of these plates has to be changed one day after plating. Change medium on day six.</p><p>EB’s can be counted here for beating percentage. In this procedure, the temperature of the medium has to be as close as possible to 37°C. Look at each EB individually with 10X magnification on the microscope. Explore the Mesoderm carefully with in each EB, if you observe three or more DISTINCT beating areas, then the EB is counted as beating. If not, the EB is counted as not beating. To obtain percentage, divide beating EB number by total EB’s counted. Do this from Day 6 to the end of experiment on Day 11 or 12 based on setup. Day 8 – Plated ES</p><p>Change Medium here</p><p>Day 10 – Plated ES</p><p>Change Medium here</p><p>Day 12 – Plated ES</p><p>IF samples are always obtained on this day</p>