TITLE: Single Cell Seeding of BBB Stem Cell Model
AUTHORS: Ethan Lippmann, Hannah Wilson, Emma Neal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Standardized single cell seeding protocol for Blood-Brain Barrier (BBB) differentiation.</div></div>

[STEPS]
?. [Reagent Preparation]
ROCK inhibitor:Make   working stock solution by diluting    into   . Use at 1:1000 for   final concentration. Aliquots can be stored long term at   for up to 1 year and frozen/thawed as many times as necessary. E8 mediaMake  working stock solution by diluting   into  . Use at 1:1000 for  final concentration. Aliquots can be stored long term at  for up to 1 year and frozen/thawed as many times as necessary.
[ROCK inhibitor]
[ultrapure distilled water]
-80 °C
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Manually transfer spent medium to a 15 ml conical. Save  of media for every well being passaged.
1 ml
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Wash each well once with .
[PBS]
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Add  (warmed to ) to each well.
[accutase]
0 Room temperature
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Incubate at  until the cells are beginning to detach (approx.  — )
37 °C
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Using p1000, collect cells, and spray gently over surface 2 — 3x to dislodge any remaining cells.
Pipetting more than this will reduce cell viability.
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Collect cells in the 15 ml conical containing spent medium.
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Spin down cells for  —  at .
Centrifuge: 1000 33
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Aspirate media, resuspend cells in . Thoroughly triturate 2 — 3 times using p1000 to yield single cell suspension.
[E8 medium]
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Take  of cells to count, drawing from the middle of the sample to prevent bias from settling cells. Transfer these cells to a clean microfuge tube.
10 µl
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Dilute the  of cells in  of . Transfer  of this diluted suspension to a Countess cell counting chamber; allow to sit for .
10 µl
10 µl
[Trypan blue]
10 µl
Note that this step is performed outside the hood and is NONSTERILE.
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Insert the slide into the Countess II automated cell counter. Note the calculated live cell density. You will use this density (not the total density) to calculate the number of cells needed for seeding.
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Calculate appropriate volume of cells to add to each 6-well.Typical seeding number for IMR90-4 iPSCs is between 100,000 – 120,000 cells per well.Typical seeding number for CC3 and CD12 iPSCs is 150,000 cells per well
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Resuspend cells in appropriate volume of E8 +  (2 ml/well).
[ROCK inhibitor]
If you have excess cells, discard the excess before resuspending.
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Place plate in incubator, and shake plate quickly back and forth (not swirling) to distribute cells evenly.
?. [Seeding cells for BBB differentiation using single cell seeding (Day -1)]
Approximately 24 hours later (Day 0), initiate differentiation:
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?. Add 3 ml A, then wait for 30.
?. Add 3ml A
?. wait for 30 min.