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Workflow for hESC Course

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)

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Workflow for hESC Course

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

  2. 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

  3. B) and C) Feeding Stem Cells Feed M/W/F With Neural Induction Medium Neural induction Medium (Monday AM) Harvesting for RNA (Monday) Passaging Cells onto new dishes (Tuesday AM) Forming Embryoid bodies (Tuesday PM) Amaxa Electroporation (Thursday) Manual Colony Picking (Wednesday)

  4. D) Feeding Neural Stem CellsSharing dish with group mate2 dishes per group Feed M/W/F With Neural differentiation Medium Neural Differentiation Medium (Monday AM) Harvesting cells for RNA (Monday PM) PassagingNSCs (Wednesday AM/PM) Neural Differentiation Medium (Monday AM) Harvesting cells for RNA (Monday PM) PassagingNSCs (Wednesday AM/PM)

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

  6. F) Harvesting ESCs and NSCs for RNA Neural induction Medium (Monday AM) Harvesting for RNA (Monday) Passaging Cells onto new dishes (Tuesday AM) Neural Differentiation Medium (Monday AM) Harvesting cells for RNA (Monday PM) PassagingNSCs (Wednesday AM/PM) Forming Embryoid bodies (Tuesday PM) Amaxa Electroporation (Thursday) Manual Colony Picking (Wednesday) Neural Differentiation Medium (Monday AM) Harvesting cells for RNA (Monday PM) PassagingNSCs (Wednesday AM/PM)

  7. G) Passaging Cells Neural induction Medium (Monday AM) Harvesting for RNA (Monday) Passaging Cells onto new dishes (Tuesday AM) 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 Forming Embryoid bodies (Tuesday PM) Amaxa Electroporation (Thursday) Manual Colony Picking (Wednesday) NOTE: Some of these wells, thawed or passaged may be used for flow cytometry on Friday Thawed Thawed

  8. (H) Embryoid Bodies ULA Vs. AggreWell dish Neural induction Medium (Monday AM) Harvesting for RNA (Monday) Passaging Cells onto new dishes (Tuesday AM) 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 Forming Embryoid bodies (Tuesday PM) Amaxa Electroporation (Thursday) Manual Colony Picking (Wednesday) Vs

  9. (I) Feeding hESCs Neural induction Medium (Monday AM) Harvesting for RNA (Monday) Passaging Cells onto new dishes (Tuesday AM) Forming Embryoid bodies (Tuesday PM) Amaxa Electroporation (Thursday) Manual Colony Picking (Wednesday)

  10. (J) Lentivirus Packaging ‐ 6cm dish HEK293FT Cells 1 million per 6 cm dish transfect with 3 plasmids to package lentivirus – observe fluorescence

  11. K) Passaging Neural Stem CellsSharing dish with group mate2 dishes per group Neural Differentiation Medium (Monday AM) Harvesting cells for RNA (Monday PM) PassagingNSCs (Wednesday AM/PM) Neural Differentiation Medium (Monday AM) Harvesting cells for RNA (Monday PM) PassagingNSCs (Wednesday AM/PM)

  12. L) Manual colony picking Original Dish Before you plate cells on DISH E, you must feed it! Use one well from original dish. Pick 10 -20 colonies and replate onto empty well of the MG coated dish (A) Neural induction Medium (Monday AM) Harvesting for RNA (Monday) Passaging Cells onto new dishes (Tuesday AM) Forming Embryoid bodies (Tuesday PM) Amaxa Electroporation (Thursday) Manual Colony Picking (Wednesday) Thawed Thawed (A) New Dish split split split

  13. 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.

  14. 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

  15. O) Electroporation of hESCs onto a 96 well dish Original 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. Neural induction Medium (Monday AM) Harvesting for RNA (Monday) Passaging Cells onto new dishes (Tuesday AM) Forming Embryoid bodies (Tuesday PM) Amaxa Electroporation (Thursday) Manual Colony Picking (Wednesday)

  16. 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

  17. 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

  18. 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 picked Thawed Thawed passaged passaged passaged Fixed cells may also be provided to you to speed up process

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

  20. T) Analyze ImmunostainingOlympus and EVOSAlso have Zeiss Wide Field and Confocal

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