IVM AND ACTIVATE PROCESS Cellular reprograming laboratory Prepare for IVM Collect oocytes CONTENT Wash and In Vitro Maturation Prepare for Activate • Remove Activate Prepare for IVM • Prepare petri for IVM before the ovaries are transported about hours • Make IVM medium following formula (TCM 199, 10% FBS, 10% follicle fluid, hCG 0.1 IU/mL, sodium pyruvate) • Prepare petri include: Floating drop: Hepes medium I II - Make 50 µl IVM medium drop - Cover with oil TCM medium IVM medium - Pump 150 µl IVM medium on top of 50 µl IVM drop Write on the lib each petri: my name+ name of medium +day/month place all of them in warm incubator Collect oocytes • • • • Washing ovaries with time of PBS Dissect follicles with diameter from to mm Washing with PBS Then, Peeling off the cell layer and immediately place them in the hepes medium • Make a pick needle by heating Pasteur pipette • Break follicles and collect oocyte in slide (?) • Using pick needle to pick oocytes in to another hepes petri Wash and In Vitro Maturation • Made a wash needle (it should be smaller than the pick needle and large enough that it won't hurt the cumulus) • Collect good cumulus oocyte complexes (COCs), then wash in drop of hepes, TCM drop and IVM medium • Transfer oocytes to drop floating (maximum about 15 oocytes each drop) • Take a picture and record time, then incubate in humidified environment, 5% CO2, at 38.5 oC Prepare for Activation • After 39-40h of IVM culture, remove cumulus from COCs • Prepare for cumulus cell removal: Cover with aluminum foil hepes CB50 + 20mg BTA Hepes dish for removing cumulus cells Recover medium (using IVM medium) CB50 + 20mg BTA (?) CB petri (NCSU medium, 1% CB, 1% hypotaurine) IVD petri (NCSU, 1% hypotaurine) • Write on the lib each petri: my name+ name of medium place all of them in warm incubator except CB50 + 20mg BTA place in the refrigerator Remove • After petri for remove have warmed (bout 30 minutes): • Prepare: a stopwatch, some needle for remove, hyaluronidase • Start timer for remove • Mix well 0.5 µl hya in a drop of IVM • Transfer oocyte to drop of recover petri of remove, then using big to small needle to remove each layer of the cumulus until completely clean • When oocytes completely lean, oocytes will be transferred to Recover petri part I • Do again with other oocytes in drop of IVM but transfer them to recover petri part II • Take a picture and gently place in incubator Activate • After 42 hours from occytes are transfer to drop floating • Made needle • Prepare chamber with a mount of medium in 100V • The oocytes will be washed through drop of medium • Place oocytes to chamber (each time about 10 eggs) • Washing oocytes in recover petri • Then washing in CB drops and put the oocytes in drop remain of CB petri • Take a picture then warm in incubator • After hours, transfer oocytes to IVD medium and IVD medium is changed at day •Thanks you for your attention