TITLE: Generation of iPSC-derived dopaminergic neurons
AUTHORS: Julie Jerber, James Haldane, Juliette Steer, Daniel Pearce, Minal Patel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes a method for the production of dopaminergic neurons from human iPSCs, using a 52-day long differentiation method adapted from </span><a href="https://doi.org/10.1038/nature10648" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">doi.org/10.1038/nature10648</span></a><span>. </span></div><div class = "text-block"><span style = "font-style:italic;">Steps in this protocol assume the use of a single 12 well plate for the purposes of differentiation; adjust volumes accordingly depending on labware used.</span></div></div>

[STEPS]
?. [Seeding iPSCs for neural induction (Day -1)]
Aspirate the media from the well(s) of the iPSC culture and add  of DPBS (-/-) to wash.
2 mL
?. [Seeding iPSCs for neural induction (Day -1)]
Aspirate the DPBS (-/-) and add  of warm Accutase to each well.
1 mL
?. [Seeding iPSCs for neural induction (Day -1)]
Incubate cells for  at
37 °C
?. [Seeding iPSCs for neural induction (Day -1)]
Inspect the cultures after incubation to confirm cell detachment.If cells are still partially adhered, incubate for a further 3 minutes.
?. [Seeding iPSCs for neural induction (Day -1)]
Add  of E8-ROCKi to each well and repeatedly wash the media over the well(s) (with a P1000 pipette) to detach the cells and dissociate them to a single-cell suspension. Confirm a majority single cell suspension has been obtained under the microscope before proceeding to the next step. If there are a lot of cell aggregates still present then triturate the cell suspensions further with a pipette. Alternatively, centrifuge the cell suspension at 250xg for 3 minutes and resuspend the cell pellet with 1mL E8-ROCKi using a P1000 pipette.
2 mL
?. [Seeding iPSCs for neural induction (Day -1)]
Collect the cell suspension from each dissociated well and transfer into 15mL centrifuge tube(s) capped with a 40µm mini cell strainer.
?. [Seeding iPSCs for neural induction (Day -1)]
Centrifuge cells at .
Centrifuge: 250 34, 3 min
?. [Seeding iPSCs for neural induction (Day -1)]
Note: These next steps describe a 1/10 dilution to prepare a 500uL suspension for cell counting with a Nucleocounter. Adjust as necessary for your chosen method of counting.Prepare a 1.5mL tube for cell counting. Add  E8-ROCKi to the tube.
450 µl
?. [Seeding iPSCs for neural induction (Day -1)]
Remove supernatant and gently re-suspend the cell pellet(s) in  of E8-ROCKi medium with a P1000 pipette. Pipette up and down several times to ensure a single cell suspension is created.If there are mutiple tubes then combine the cell suspensions together in one tube. Top up to   with E8-ROCKi and mix well.
1 mL
5 mL
?. [Seeding iPSCs for neural induction (Day -1)]
Immediately transfer  of the re-suspended cell suspension into the 1.5mL tube prepared in step 15 and perform a viable cell count.
50 µl
?. [Seeding iPSCs for neural induction (Day -1)]
Prepare the Geltrex-coated 12 well plate for seeding by aspirating excess Geltrex and adding  of E8-ROCKi into each coated well to be used. Do not let the coated wells dry out.
1 mL
?. [Seeding iPSCs for neural induction (Day -1)]
Thoroughly mix and resuspend the cell suspension prepared in step 18 by pipetting up and down with a stripette and then transfer  to each well of the 12 well plate.
1 mL
?. [Seeding iPSCs for neural induction (Day -1)]
Transfer the plate to a tissue culture incubator set at   and 5% CO2.  Agitate plate gently on the incubator shelf to ensure even distribution of cells across wells and then leave undisturbed overnight.
37 °C
?. [Media Composition]
Base Media:Listed below are the reagent quantities needed for composition of the three types of base media used throughout this protocol. All of these base media require further supplements prior to media change; supplements needed are listed in the Neural Induction Media and Maturation Medium steps.Note: Use-within dates are only for the base media, not for media with added supplements. Supplemented media (i.e. Neural Induction and Maturation media) should only be used on the day of preparation.  ABCD1KOSR (use within 1 week)100mL1mL[Final]2Knockout DMEM81.98 mL819.8 µL-3Knockout Serum Replacement (KSR)15 mL150 µL15%4Glutamax (100X)1 mL10 µL1X5MEM Non Essential Amino Acids (NEAA) (100X)1 mL10 µL1mM62-mercaptoethanol (50mM)20 µL0.2 µL0.01mM7Penicillin-Streptomycin (10,000 U/mL)1 mL10 µL100 U/mL  ABCD1NNB (Use within 3 days)100mL1mL[Final]2Neurobasal medium96.5 mL965 µL-3N2 (100X)0.5 mL5 µL0.5X4B27 (50X)1 mL10 µL0.5X5Glutamax (100X)1 mL10 µL1X6Penicillin-Streptomycin (10,000 U/mL) 1 mL10 µL100 U/mL  ABCD1NB/B27 (Use within 3 days)100mL1mL[Final]2Neurobasal medium96 mL0.96 mL-3B27 (50X)2 mL20 µL1X4Glutamax (100X)1 mL10 µL1X5Penicillin-Streptomycin (10,000 U/mL)1 mL10 µL100 U/mL
ABCD1KOSR (use within 1 week)100mL1mL[Final]2Knockout DMEM81.98 mL819.8 µL-3Knockout Serum Replacement (KSR)15 mL150 µL15%4Glutamax (100X)1 mL10 µL1X5MEM Non Essential Amino Acids (NEAA) (100X)1 mL10 µL1mM62-mercaptoethanol (50mM)20 µL0.2 µL0.01mM7Penicillin-Streptomycin (10,000 U/mL)1 mL10 µL100 U/mL
ABCD1NNB (Use within 3 days)100mL1mL[Final]2Neurobasal medium96.5 mL965 µL-3N2 (100X)0.5 mL5 µL0.5X4B27 (50X)1 mL10 µL0.5X5Glutamax (100X)1 mL10 µL1X6Penicillin-Streptomycin (10,000 U/mL) 1 mL10 µL100 U/mL
ABCD1NB/B27 (Use within 3 days)100mL1mL[Final]2Neurobasal medium96 mL0.96 mL-3B27 (50X)2 mL20 µL1X4Glutamax (100X)1 mL10 µL1X5Penicillin-Streptomycin (10,000 U/mL)1 mL10 µL100 U/mL
?. [Media Composition]
Maturation Medium:This is the medium that will be used for the majority of this protocol (Day 13 onward). The complete supplemented medium must be used only on the day of preparation, though the base medium (NB/B27) can be prepared earlier and used within 3 days.  ABCD1Maturation Medium (Use within 1 day)28mL1mL[Final]2NB/B273Neurobasal medium26.88 mL960 µL-4B27 (50X)560 µL20 µL1X5Glutamax (100X)280 µL10 µL1X6Penicillin-Streptomycin (10,000 U/mL)280 µL10 µL100 U/mL7Supplements8BDNF (stock 100 µg/mL)5.6 µL0.2 µL20 ng/mL9DAPT (stock 50 mM)5.6 µL0.2 µL10 uM10GDNF (stock 100 µg/mL)5.6 µL0.2 µL20 ng/mL11TGFB3 (stock 25 µg/mL)1.12 µL0.04 µL1 ng/mL12Dibutyryl cAMP (stock 100mM)140 µL5 µL0.5 mM13Ascorbic acid  (stock 200mM)28 µL1 µL0.2 mM
ABCD1Maturation Medium (Use within 1 day)28mL1mL[Final]2NB/B273Neurobasal medium26.88 mL960 µL-4B27 (50X)560 µL20 µL1X5Glutamax (100X)280 µL10 µL1X6Penicillin-Streptomycin (10,000 U/mL)280 µL10 µL100 U/mL7Supplements8BDNF (stock 100 µg/mL)5.6 µL0.2 µL20 ng/mL9DAPT (stock 50 mM)5.6 µL0.2 µL10 uM10GDNF (stock 100 µg/mL)5.6 µL0.2 µL20 ng/mL11TGFB3 (stock 25 µg/mL)1.12 µL0.04 µL1 ng/mL12Dibutyryl cAMP (stock 100mM)140 µL5 µL0.5 mM13Ascorbic acid  (stock 200mM)28 µL1 µL0.2 mM
?. [Media Composition]
Neural Induction Media:This is the media that will be used for the neural induction stage (Day 0-12). Each complete medium must be used only on the day of preparation, though the unsupplemented base media (varies) can be prepared earlier and used within the associated timespan listed in the 'Base Media' tables.During neural induction, the media is gradually altered from 100% KOSR to 25% KOSR/75% NNB.Days listed in the table assume that iPSC seeding (Day -1) takes place on a Friday.  ABCDE1Day 0Neural Induction Medium-128 mL1mL[Final]2(Sat)KOSR28mL1 mL100%3LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM4SB431542 (stock 10 mM)28 µL1 µL10 µM5Day 1-2Neural Induction Medium-228 mL1 mL6(Sun/Mon)KOSR28mL1 mL100%7LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM8SB431542 (stock 1 0mM)28 µL1 µL10 µM9SAG (stock 1 mM) 2.8 µL0.1 µL100 nM10Purmorphamine (stock 20 mM)2.8 µL0.1 µL2 µM11FGF8a (stock 100 µg/mL)28 µL1 µL100ng/mL12Day 3-4Neural Induction Medium-328 mL1 mL[Final]13(Tue/Wed)KOSR28mL1 mL100%14LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM15SB431542 (stock 10 mM)28 µL1 µL10 µM16SAG (stock  1 mM) 2.8 µL0.1 µL100 nM17Purmorphamine (stock 20 mM)2.8 µL0.1 µL2 µM18FGF8a (stock 100 µg/mL)28 µL1 µL100ng/mL19CHIR99021 (stock 10 mM)8.4 µL0.3 uL3 µM20Day 5-6Neural Induction Medium-428 mL1 mL[Final]21(Thu/Fri)KOSR21 mL0.75 mL75%22NNB7 mL0.25 mL25%23LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM24SAG (stock 1mM) 2.8 µL0.1 µL100 nM25Purmorphamine (stock 20mM)2.8 µL0.1 µL2 µM26FGF8a (stock 100 µg/mL)28 µL1 µL100ng/mL27CHIR99021 (stock 10 mM)8.4 µL0.3 uL3 µM28Day 7-8Neural Induction Medium-528 mL1 mL[Final]29(Sat/Sun)KOSR14 mL0.5 mL50%30NNB14 mL0.5 mL50%31LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM32CHIR99021 (stock 10 mM)8.4 µL0.3 uL3 µM33Day 9-10Neural Induction Medium-628 mL1 mL[Final]34(Mon/Tue)KOSR7 mL0.25 mL25%35NNB21 mL0.75 mL75%36LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM37CHIR99021 (stock 10 mM)8.4 µL0.3 uL3 µM38Day 11-12Neural Induction Medium-728 mL1 mL[Final]39(Wed/Thu)Maturation Medium28 mL1 mL100%40CHIR99021 (stock 10 mM)8.4 µL0.3 µL3 µM
ABCDE1Day 0Neural Induction Medium-128 mL1mL[Final]2(Sat)KOSR28mL1 mL100%3LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM4SB431542 (stock 10 mM)28 µL1 µL10 µM5Day 1-2Neural Induction Medium-228 mL1 mL6(Sun/Mon)KOSR28mL1 mL100%7LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM8SB431542 (stock 1 0mM)28 µL1 µL10 µM9SAG (stock 1 mM) 2.8 µL0.1 µL100 nM10Purmorphamine (stock 20 mM)2.8 µL0.1 µL2 µM11FGF8a (stock 100 µg/mL)28 µL1 µL100ng/mL12Day 3-4Neural Induction Medium-328 mL1 mL[Final]13(Tue/Wed)KOSR28mL1 mL100%14LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM15SB431542 (stock 10 mM)28 µL1 µL10 µM16SAG (stock  1 mM) 2.8 µL0.1 µL100 nM17Purmorphamine (stock 20 mM)2.8 µL0.1 µL2 µM18FGF8a (stock 100 µg/mL)28 µL1 µL100ng/mL19CHIR99021 (stock 10 mM)8.4 µL0.3 uL3 µM20Day 5-6Neural Induction Medium-428 mL1 mL[Final]21(Thu/Fri)KOSR21 mL0.75 mL75%22NNB7 mL0.25 mL25%23LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM24SAG (stock 1mM) 2.8 µL0.1 µL100 nM25Purmorphamine (stock 20mM)2.8 µL0.1 µL2 µM26FGF8a (stock 100 µg/mL)28 µL1 µL100ng/mL27CHIR99021 (stock 10 mM)8.4 µL0.3 uL3 µM28Day 7-8Neural Induction Medium-528 mL1 mL[Final]29(Sat/Sun)KOSR14 mL0.5 mL50%30NNB14 mL0.5 mL50%31LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM32CHIR99021 (stock 10 mM)8.4 µL0.3 uL3 µM33Day 9-10Neural Induction Medium-628 mL1 mL[Final]34(Mon/Tue)KOSR7 mL0.25 mL25%35NNB21 mL0.75 mL75%36LDN193189 (stock 1 mM)2.8 µL0.1 µL100 nM37CHIR99021 (stock 10 mM)8.4 µL0.3 uL3 µM38Day 11-12Neural Induction Medium-728 mL1 mL[Final]39(Wed/Thu)Maturation Medium28 mL1 mL100%40CHIR99021 (stock 10 mM)8.4 µL0.3 µL3 µM
?. [Seeding iPSCs for neural induction (Day -1)]
Seeding iPSCs for Neural Induction (Day -1):This section covers the seeding of iPSCs onto a prepared 12 well Geltrex-coated plate for neural induction.Note: Our lab carried out Day -1 steps on a Friday and any days mentioned in the protocol assume this to be the case.Cells are seeded at a density of 200,000 cells/cm2 or 760,000 cells per well in a 3.8cm2 12 well. The total number of cells required to seed a 12 well plate is at least 9.2x106 cells. Select an adequate number of high quality iPSC wells/ plates/ flasks at a suitable density for harvesting. As a general guide a 60-80% confluent well on a 6 well plate (9.6cm2) should yield 2-4 million cells.
?. [Plate Preparation (Day -2 & 19)]
Preparing Geltrex-coated Plate(s) (Day -2 & 19):This section covers the preparation of the 12 well Geltrex-coated plate(s) that:iPSCs will be seeded onto for neural induction (Day -1)Early neural progenitors will be seeded onto for neuronal maturation (Day 20)We recommend preparing the coated plates 1 day prior to the day of use.Prepare 10% Geltrex stock aliquots:Thaw a vial of Geltrex at   overnight.Aliquot into sterile tubes in single use volumes. Ensure the temperature does not exceed  while working with Geltrex.Store aliquots at Prior to coating plates, thaw 10% Geltrex aliquots at  overnight or on the day required.
4 °C
15 °C
-80 °C
4 °C
?. [Plate Preparation (Day -2 & 19)]
Prepare 1% Geltrex by adding  of 10% Geltrex to  of ice-cold DMEM:F12 w/o phenol red, keeping the Geltrex on ice during the process. Note: if lumps are observed in the Geltrex aliquot then it should not be used (to avoid premature gelling).
1.25 mL
11.25 mL
?. [Plate Preparation (Day -2 & 19)]
Add  of cold 1% Geltrex to each well of the 12 well plate.
1 mL
?. [Plate Preparation (Day -2 & 19)]
Incubate the plate at  for at least , but not more than 24 hours. Equilibrate plates to room temperature for 1 hour before use.
37 °C
?. [Seeding iPSCs for neural induction (Day -1)]
Prepare E8-ROCKi medium by supplementing E8 with ROCK inhibitor (Y-27632) to a final concentration of 10μM.
?. [Seeding iPSCs for neural induction (Day -1)]
Prepare a cell suspension of 9.9x106 viable iPS cells in  E8-ROCKi. This is enough to seed 12 wells at a seeding density of 760,000 cells/well (or 2x105 cells/cm2). If seeding a different number of wells always prepare a slightly higher volume than required to account for volume loss during processing.
13 mL
?. [Media Change (Day 0-52)]
Media Change (Day 0-52):Confirm how much media you will need for the next few days before making up base media (or for the day in the case of supplemented media).Base media should be warmed to between   and   before adding supplements. Do not put complete supplemented media in the water bath.Perform media change very gently, especially later on into the differentiation process, as cells can detach very easily. We recommend tilting plates and dispensing media down the side of each well to reduce force applied to the cell layer.Refer to the 'Media Composition' section for details on each media type and which media to use for Day 0-12.  ABCD1DayMedia ChangeMedia TypeMedia feed volume per well20-12DailyDiffers (See 'Media Composition')2 mL313-19DailyMaturation Medium2 mL420PassageMaturation Medium-521-22DailyMaturation Medium1 mL623-33Every other dayMaturation Medium2 mL734-52Every 2 (*or 3) daysMaturation Medium2mL (*or 3mL)
0 Room temperature
37 °C
ABCD1DayMedia ChangeMedia TypeMedia feed volume per well20-12DailyDiffers (See 'Media Composition')2 mL313-19DailyMaturation Medium2 mL420PassageMaturation Medium-521-22DailyMaturation Medium1 mL623-33Every other dayMaturation Medium2 mL734-52Every 2 (*or 3) daysMaturation Medium2mL (*or 3mL)
?. [Passage (Day 20)]
Passaging of Neuro-epithelial sheet (Day 20):This section covers the passaging of the neuro-epithelial sheet on Day 20. Cells are lifted and re-seeded for neuronal maturation at 3.5x105 cells/cm2 on a 12 well Geltrex-coated plate (prepare prior to these steps as described in the 'Plate Preparation' section). This is the only passage during the differentiation process.Note: These steps assume seeding back onto one 12 well plate, however we found that we consistently had more than enough cells to seed two 12 well plates after this passage; adjust volumes as needed.
?. [Seeding iPSCs for neural induction (Day -1)]
Neural Induction (Day 0):Inspect the plate(s) and verify that the cells are well-distributed and ~90-100% confluent.From this point, carry out a daily media change as described in the 'Media Change' section until Day 20 when the culture is passaged.
?. [Supplement Preparation]
Supplement Preparation:Supplements to be added to media (on day of use) in this protocol are listed in the table below, with recommended stock concentrations for aliquots, concentrations to be used on cells and the diluent needed for each reagent. It is recommended to aliquot supplements in volumes for single use. Aliquots should be stored at  .  ABCD1SupplementRecommended Stock ConcentrationFinal Concentration to be used on cellsDiluent2Ascorbic Acid200 mM0.2 mMWater for Cell Culture3hBDNF100 µg/mL20 ng/mL0.1% BSA in Water for Cell Culture4CHIR9902110 mM3 µMDMSO5DAPT50 mM10 µMDMSO6dibutyryl cAMP100 mM0.5 mMWater for Cell Culture7hFGF8a100 µg/mL100 ng/mL0.1% BSA in Water for Cell Culture8hGDNF100 µg/mL20 ng/mL0.1% BSA in Water for Cell Culture9LDN1931891 mM100 nMDMSO10Purmorphamine20 mM1 µMDMSO11SAG1 mM100 nMDMSO12SB43154210mM10µMDMSO13TGF-β325µg/mL1ng/mL10mM Citric Acid & 0.1% BSA in Water for Cell Culture (1:3)*10% BSA stock solution (100 mg/mL): Dissolve Bovine Serum Albumin powder in sterile Water for Cell Culture at 100 mg/mL. Filter sterilise with a 0.2 µm sterilising grade filter. Aliquot into sterile microcentrifuge tubes. Store at -20 °C. Thaw at room temperature and resuspend prior to use.0.1% BSA Solution (1mg/mL):Dilute a thawed aliquot of 10% BSA solution 100-fold with sterile Water for Cell Culture to create a 0.1% BSA solution; e.g. add 100µL 10% BSA to 9.9mL water.
-80 °C
ABCD1SupplementRecommended Stock ConcentrationFinal Concentration to be used on cellsDiluent2Ascorbic Acid200 mM0.2 mMWater for Cell Culture3hBDNF100 µg/mL20 ng/mL0.1% BSA in Water for Cell Culture4CHIR9902110 mM3 µMDMSO5DAPT50 mM10 µMDMSO6dibutyryl cAMP100 mM0.5 mMWater for Cell Culture7hFGF8a100 µg/mL100 ng/mL0.1% BSA in Water for Cell Culture8hGDNF100 µg/mL20 ng/mL0.1% BSA in Water for Cell Culture9LDN1931891 mM100 nMDMSO10Purmorphamine20 mM1 µMDMSO11SAG1 mM100 nMDMSO12SB43154210mM10µMDMSO13TGF-β325µg/mL1ng/mL10mM Citric Acid & 0.1% BSA in Water for Cell Culture (1:3)*
?. [Passage (Day 20)]
Prepare 30mL of Wash Buffer 1 and 12mL Wash Buffer 2 for every 12 well plate to be dissociated.
?. [Passage (Day 20)]
Prepare Seeding Medium by supplementing  of Maturation Medium with ROCK inhibitor (Y-27632) to a final concentration of 10μM.
30 mL
?. [Passage (Day 20)]
Prepare Dissociation Buffer by supplementing  Accutase (warmed) with 2 vials Papain (PDS Kit). Reconstitute the lyophilised papain with the Accutase, replace the lid on the vial and invert several times ensuring all powder in the vial and on the lid is dissolved. Transfer solution back into the Dissociation Buffer tube.0.5mL Dissociation Buffer is required per well to be harvested. 5 wells of a 12 well plate (2.5mL Dissociation Buffer) will provide enough cells to seed onto one 12 well plate; adjust volumes as needed.
10 mL
?. [Passage (Day 20)]
Aspirate the media from the well(s) of the neuronal culture and gently add  of DPBS (-/-) without disturbing the cell layer.
1 mL
?. [Passage (Day 20)]
Aspirate the DPBS (-/-) and add  of Dissociation Buffer to each well.
0.5 mL
?. [Passage (Day 20)]
Transfer cells to a  5% CO2 tissue culture incubator. Incubate for .
37 °C
?. [Passage (Day 20)]
Complete the preparation of Wash Buffer 1 within the last 5 minutes of incubation of the cells. Retrieve the DNase (D2) vial(s) from  storage. Reconstitute the vial(s) by adding  Wash Buffer 1 to each vial to make a  solution. Make sure to reconstitute all the powder (check the lid). Avoid vigorous mixing - do not vortex.
4 °C
250 µl
?. [Passage (Day 20)]
Following incubation inspect the cells under a microscope. The cells should be detaching from the plate and the cell layer should have a darkened appearance. Cells will have a rounded appearance as they dissociate and individual cells should be visible at the edges or in gaps in the cell layer.
?. [Passage (Day 20)]
Optional: Test for ease of dissociation by gently pipetting ~ of the Dissociation Buffer against the cells with a P1000 pipette. If cells do not dissociate easily, extend digestion by  and repeat this step, but do not exceed 30 mins.
100 µl
?. [Passage (Day 20)]
Add  of Wash Buffer 1 per well.
2 mL
?. [Passage (Day 20)]
Repeatedly wash the buffer over the well(s) with a P1000 pipette to detach the cells and dissociate them to a single-cell suspension. Sufficient dissociation is critical at this step. It is recommended to check the cell suspension under a microscope and if necessary pipette the cell suspension up and down further until the majority is single cells. Try to avoid over-pipetting the cells as this could affect viability.
?. [Passage (Day 20)]
Transfer the cell suspension into a 15mL centrifuge tube capped with a 40µm cell strainer.
?. [Passage (Day 20)]
Optional: If there are residual cells in the plate, wash well(s) with  of Wash Buffer 1 and transfer this suspension into the tube from the previous step.
0.5 mL
?. [Passage (Day 20)]
Centrifuge cells at
Centrifuge: 150 34, 3 min
?. [Passage (Day 20)]
Aspirate supernatant and gently re-suspend the cell pellet in  (~1mL per well harvested) of Wash Buffer 2.
12 mL
?. [Passage (Day 20)]
Centrifuge cells at .
Centrifuge: 150 34, 3 min
?. [Passage (Day 20)]
Aspirate supernatant and gently re-suspend the cell pellet in  of Seeding Medium.
5 mL
?. [Passage (Day 20)]
Immediately transfer  of the re-suspended cell suspension into the 1.5mL tube prepared in step 44 and perform a viable cell count.
50 µl
?. [Passage (Day 20)]
Prepare a cell suspension of 1.729x107 viable cells in  Seeding Medium. This is enough to seed 12 wells at a density of 1.33x106 cells per well. If seeding a different number of wells always prepare a slightly higher volume than required to account for volume loss during processing.
13 mL
?. [Passage (Day 20)]
Aspirate excess Geltrex from the prepared 12 well plate and immediately add  of Seeding Medium into each coated well to be used. Do not let the coated wells dry out.
1 mL
?. [Passage (Day 20)]
Thoroughly mix and resuspend the cell suspension prepared in step 47 by pipetting up and down with a stripette. Transfer  of the cell suspension to each well of the prepared Geltrex 12 well plate.
1 mL
?. [Passage (Day 20)]
Transfer the plate to a tissue culture incubator set at   and 5% CO2.  Agitate the plate gently on the incubator shelf to ensure even distribution of cells across wells and then leave undisturbed overnight.
37 °C
?. [Passage (Day 20)]
Note: These next steps describe a 1/10 dilution to prepare a 500uL suspension for cell counting with a Nucleocounter. Adjust as necessary for your chosen method of counting.Prepare a 1.5mL tube for cell counting. Add  Wash Buffer 2 to the tube.
450 µl
?. [Passage (Day 20)]
On Day 21 inspect the plate(s) and verify that the cells are attached and well distributed.Continue to maintain the culture with media changes as described in the 'Media Change' section up to Day 52 (or beyond, according to your requirements)
?. [Passage (Day 20)]
Supplement  DMEM:F12 + GlutaMAX with ROCK inhibitor (Y-27632) to a final concentration of .
42 mL
?. [Passage (Day 20)]
Split this into two tubes of  (for Wash Buffer 1) and  (Wash Buffer 2).Wash Buffer 1 will be further supplemented with DNase1 in step 34.1 vial DNase (D2) should be added for every 15mL of Wash Buffer 1 prepared. However, DNase should only be added immediately prior to use of the buffer when the cells are nearing the end of their dissociation. Keep the DNase at 4 °C until use.
30 mL
12 mL