TITLE: An Optimized Protocol for the Generation of Cortical Neurons from human iPSCs under Defined Conditions
AUTHORS: Clare L Parish, Lachlan L Thompson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about cortical differentiation.</div></div>

[STEPS]
?. [Day -1:]
Coat plastic cell culture plate (laminin 521 ).Make single cells of hPSCs using accutase for - @ .Seed cells @ 375K/cm2.
37 °C
?. [d0:]
mTESR1 media change ~1-2 hrs (up to 6 hrs) prior to differentiation initiation.
?. [d0:]
Wash cells with PBS-/- ( for 48 wells,  for 96 wells).
500 µl
200 µl
?. [d0:]
Add  to initiate differentiation.
[ d0 media]
?. [d0-11: Daily media change]
[CTX Base supplemented with SB (1:1000) LDN (1:2500)]
?. [d11: Passage and fix]
4 wells will be passaged 1:2.5 (10 in total) into 8 new hLaminin-coated wells.Remaining 2 wells will be fixed for staininghLaminin coat 8x 48 wells for passaging and allow > for coating
?. [d11: Passage]
Aspirate media and wash wells with .
[PBS-/-]
?. [d11: Passage]
Add  to each well and incubate for ~-.
[EDTA]
?. [d11: Passage]
When clumps are ready to detach, collect into a 15 ml tube with .Important note: in these stages of EDTA digestion, cells will be in clumps/rosettes, do not over-triturate suspension as this will break up cells resulting in poor rosette formation.
[PBS-/-]
?. [d11: Passage]
Wash each well again with 1x to ensure all cells have been collected.
[EDTA]
?. [d11: Passage]
Centrifuge at .
Centrifuge: 1100 33, 3 min
?. [d11: Passage]
Aspirate supernatant and gently flick pellet.
?. [d11: Passage]
Pipette  gently to get clumps/cells in suspension.
[CTX base with SB/LDN + Ri (1:1000)]
DO NOT vigorously pipette to single cell. Sometimes just the addition of the 1 ml is enough to distribute cells/clumps.
?. [d11: Passage]
Add remaining CTX base with SB/LDN + Ri (1:1000) volume for  cell seeding.
?. [d11: Passage]
Aspirate hLam from new plate and add .
[CTX base with SB/LDN + Ri  (1:1000)]
?. [d11: Passage]
Pipette  into each well.
[cell suspspension]
?. [d11: Passage]
Ensure even distribution by pipetting the peace sign gently.
?. [d11: Passage]
Allow clumps to attach, check on microscope and place gently into incubator.
?. [d12: Media change every 2nd day]
Aspirate the CTX media and refresh with
[CTX base supplemented with FGF2 (1:5000)]
?. [d19: Passage and fix]
5 wells will be passaged into 1:2.5; 10x 48 wells for maintenance  Remaining 2.5 wells will be fixed for staininghLam coat 13x 48 wells for passaging and allow > for coating NB: At this point in the CTX differentiation protocol, a significant amount of progenitor expansion will have occurred in response to FGF2 exposure. Typically, a 5/6-fold increase in the cell numbers in achieved from Day 0 seeding. Cell numbers from 1 well of a 48-well plate can yield between 1.5-2.0 x 106 neural progenitors. Cells seeded at 4.5 x 105 cells/cm2. Coat the required number of wells depending on specific application.
?. [d19:Passage]
Aspirate media and wash wells with .
[PBS-/-]
?. [d19:Passage]
Add  to each well and incubate for ~-.
[Accutase]
?. [d19:Passage]
When cells are ready to detach, collect into a 15 ml tube with .
[PBS-/-]
?. [d19:Passage]
Wash each well again with 1x  to ensure all cells have been collected.
[PBS-/-]
?. [d19:Passage]
Centrifuge at .
Centrifuge: 1400 33, 3 min
?. [d19:Passage]
Aspirate supernatant and gently flick pellet.
?. [d19:Passage]
Pipette  gently to get cells in suspension.
[CTX base with FGF2 + Ri (1:1000)]
?. [d19:Passage]
Perform cell count and transfer required volume of cell suspension for 4.5 x 105cell/cm2 into a fresh centrifuge tube containing CTX base media supplemented with ROCKi (10 µM) and FGF2 (20 ng/ml).
?. [d19:Passage]
Aspirate hLam from new plate and add .
[CTX base with FGF2 + Ri  (1:1000)]
?. [d19:Passage]
Pipette  into each well.
[cell suspspension]
?. [d19:Passage]
Ensure even distribution by pipetting the peace sign gently.
?. [d19:Passage]
Allow cells to attach, check on microscope and place gently into incubator.
?. [d20: Media change every 2nd day]
Media change with .
[CTX base (no factors or small molecules)]
?. [d25: Passage and fix]
hLam coat wells for passaging and allow > for coating
?. [d25: Passage]
Aspirate media and wash wells with .
[PBS-/-]
?. [d25: Passage]
Add  to each well and incubate for ~-.
[Accutase]
?. [d25: Passage]
Add   to each well and transfer cell suspension into a clean 15 ml centrifuge tube.
[CTX media]
?. [d25: Passage]
Centrifuge at .
Centrifuge: 1400 33, 3 min
?. [d25: Passage]
Aspirate media and flick tube to dislodge pellet. Resuspend cells in (at least 5 ml) CTX base media supplemented with ROCKi.
?. [d25: Passage]
Aspirate hLam from new plate and add .
[cell suspension in CTX base media supplemented with ROCKi]
?. [d25: Passage]
Ensure even distribution by moving the plate in a ‘figure 8’ pattern.
?. [d25: Passage]
Allow cells to attach, check on microscope and place gently into incubator.
?. [d26: Media change every 2nd day]
?. [Fix:]
Wash wells with 200 μL PBS-/-.
?. [Fix:]
Add  for .
[4% PFA]
?. [Fix:]
Aspirate PFA.
?. [Fix:]
Wash 3x with PBS-/-.
?. [Fix:]
Wash with PBS-/-.
?. [Fix:]
Fill wells with PBS/Azide or begin staining.
?. [Fix:]
Wash with PBS-/-.
?. [Fix:]
Wash with PBS-/-.
?. [Staining:]
Permeabilize cells with - for -.
200 µl
[PADT]
?. [Staining:]
Aspirate PADT and pipette on primary antibodies to incubate  at .
4 °C
?. [Staining:]
Aspirate primaries.
?. [Staining:]
Wash wells 2x with PBS-/-.
?. [Staining:]
Wash wells with PBS-/-.
?. [Staining:]
Wash wells with PBS-/-.
?. [Staining:]
Block with  for .
[PADT]
?. [Staining:]
Aspirate PADT and pipette on secondaries to incubate for - at room temperature.
?. [Staining:]
Aspirate secondaries and wash 2x with .
[PBS-/-]
?. [Staining:]
Wash with .
[PBS-/-]
?. [Staining:]
Wash with .
[PBS-/-]
?. [Staining:]
DAPI stain for -.
?. [Staining:]
Wash DAPI with 1-2x PBS-/- wash.
?. [Staining:]
Wash DAPI with PBS-/- wash.
?. [Staining:]
Optional: Wash DAPI with PBS-/- wash.
?. [Staining:]
Fill well with PBS/Azide for long-term storage.
?. [Staining:]
Image.
?. [d29: prepare coverslips for passaging]
Remove sterile coverslips from Ethanol using sterile tweezers.
?. [d29: prepare coverslips for passaging]
Place coverslips into 48-well plates and wash x 3 with 500 μL PBS-/-, with 5 min incubations, at Room temperature to remove all residual Ethanol.
?. [d30: passage]
Remove 48-well plate, containing PO-coated coverslips from the fridge and wash x 2 with 5-minute incubations at RT.
?. [d29: prepare coverslips for passaging]
Add 300 μL of Poly-L-Ornithine (PO) to each well and incubate overnight at 4˚C. Each well should contain a single coverslip.
?. [d30: passage]
Add 190 µl of 5 µg/ml of L521 solution diluted in 1 x PBS +/+ to each well with a coverslip and incubate for 2 hours at 37˚C.
?. [d30: passage]
Dissociate cells as performed at D25 timepoint.
?. [d30: passage]
Add 300 µl of Accutase to each of the wells and place in the incubator for 10 minutes at 37˚C.
?. [d30: passage]
Following incubation, add 300 μL of CTX media to each well, harvest cells and transfer cell suspension into a clean 15 ml centrifuge tube.
?. [d30: passage]
Centrifuge cell suspension at 1,400 rpm for 4 minutes.
?. [d37-55: maturation]
Media is aspirated and replaced with maturation media (NBB27 media) supplemented with BDNF (40 ng/ml), GDNF (40 ng/ml), dcAMP (0.05 mM), AA (200 nM) and laminin (1 μg/ml; Sigma-Aldrich).
?. [d30: passage]
Aspirate media and flick tube to dislodge pellet. Resuspend cells in (at least 5 ml) CTX base media supplemented with ROCKi, 10 µM.
?. [d37-55: maturation]
Cortical neurons are maintained in this media with growth factors until Day 55, where a final immunocytochemical analysis for cortical patterning is performed.