Workflow for hESC Course March 26 th -30 th, 2009

A) Preparing 6 well Matrix Coated Dish Coating with Matrigel (Monday AM) and will be used for: (E) plating a thawed vial of hESCs onto (Monday PM) (G) passaging cells onto (Tuesday AM) (L) picking cells, and planting onto (Wed AM/PM) May also coat other dish with ¼ Matrigel for NSCs for Wednesday AM/PM

B) and C) Feeding Stem Cells Feed M/W/F With Neural Induction Medium

D) Feeding Neural Stem Cells Sharing dish with group mate 2 dishes per group Feed M/W/F With Neural differentiation Medium

(E) Thaw hESCs onto 2 wells of a 6 well dish which was coated in step (A)

F) Harvesting ESCs and NSCs for RNA

G) Passaging Cells Before you passage onto this dish from (A) you must feed the “thawed” wells first! You can also feed two other wells from original dish marked in blue now or later Passage one well into 2-3 wells using Dispase NOTE: Some of these wells, thawed or passaged may be used for flow cytometry on Friday

(H) Embryoid Bodies ULA Vs. AggreWell dish Passage cells from one well from (B) onto 1 well of this ULA dish or into an Aggrewell plate. You will share this dish with your group. The cells won’t stick, and will form embryoid bodies Vs

(I) Feeding hESCs

(J) Lentivirus Packaging ‐ 6cm dish

K) Passaging Neural Stem Cells Sharing dish with group mate 2 dishes per group

L) Manual colony picking Before you plate cells on DISH E, you must feed it! Use one well from original dish. Pick colonies and replate onto empty well of the MG coated dish (A) (A) New Dish Original Dish

M) Feeding hESC/NSC Since you passaged NSCs, you just need to feed the one remaining well form the original NSC dish with NDM Need to feed one well on the original hESC dish with NIM, and other one with mTeSR. You should have already fed other well on original dish and the new dish (A) before you plated via manual colony picking.

N) Immunostaining hESC/NSC Instructors will prepare immunostained slides for group, primary overnight Optional: members of group may help instructors if they finish feeding, observing and documenting their cultures. Slides will be ready to observe late Thursday, or all day Friday

O) Electroporation of hESCs onto a 96 well dish Take one well of hESCs from the 6 well dish (B) and remove cells with Accutase. Cells will be electroporated and transfected with GFP reporter. Cells will be plated into a few wells of a matrigel coated 96 well dish. Original Dish

P) Feeding hESC/NSC Feed one well with NDM – original NSCs Feed passaged NSCs with NPM New dish (A) – 6 wells with mTeSR May have cells left in one well on Original dish that you used for manual colony picking Feed EBs

Q) Immunostaining hESC/NSC Instructors will wash and use secondary antibodies, then counter-stain nuclei with DAPI Optional: members of group may help instructors if they finish feeding, observing and documenting their cultures. Slides will be ready to observe late Thursday, or all day Friday

R) Flow Cytometry You will remove cells with Accutase, stain them with directly labeled antibodies (SSEA1 and SSEA4) and load them Accuri C6 Flow Cytometers Fixed cells may also be provided to you to speed up process

S) Flow Cytometry – Analyze Nucleofection Remove cells with Accutase and analyze the GFP levels using the Accuri C6 Flow Cytometer

T) Analyze Immunostaining Olympus and EVOS Also have Zeiss Wide Field and Confocal