TITLE: iNeuron pre-differentiation & differentiation protocol
AUTHORS: Merissa Chen, Nina Draeger, Martin Kampmann, Kun Leng, Emmy Li, Connor Ludwig, Gregory Mohl, Avi Samelson, Sydney Sattler, Ruilin Tian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes thedifferentiation of iPSCs with stably integrated doxycycline-inducible Ngn2 (such as i3Ns).</div></div>

[STEPS]
?. [Culture iPSCs]
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?. [Days -3-0: Pre-differentiation]
Make N2 Pre-differentiation Media:  ABCDEF1ComponentStock ConcentrationFinal ConcentrationDilution FactorFor 100 mL (mL)For 50 mL (units in cell)2Konckout DMEM/F121X1X110050mL3NEAA100X1X1001500uL4N2 Supplement100X1X1001500uL5NT-310ug/mL10ng/mL10000.150uL6BDNF10ug/mL10ng/mL10000.150uL7Mouse Laminin*1.0mg/mL1ug/mL10000.150uL8ROCK Inhibitor**10mM10uM10000.150uL9Doxycycline***2mg/mL2ug/mL10000.150uL
Pre-Differentiation and Differentiation Notes:1. Keep reagents at   for a maximum of 4 weeks, If needing long term storage, don’t freeze-thaw more than 3 times.2. Sometimes pre-differentiated cells take a long time to come off of the plate on Day 0. This is usually okay. It is important that your cells are single cells.3. Feeding schedule and volumes:
ABCDEF1ComponentStock ConcentrationFinal ConcentrationDilution FactorFor 100 mL (mL)For 50 mL (units in cell)2Konckout DMEM/F121X1X110050mL3NEAA100X1X1001500uL4N2 Supplement100X1X1001500uL5NT-310ug/mL10ng/mL10000.150uL6BDNF10ug/mL10ng/mL10000.150uL7Mouse Laminin*1.0mg/mL1ug/mL10000.150uL8ROCK Inhibitor**10mM10uM10000.150uL9Doxycycline***2mg/mL2ug/mL10000.150uL
?. [Days -3-0: Pre-differentiation]
Coat plate with matrigel diluted in Knockout DMEM. Coat for at least  (or O/N). Coated plates can last in the  incubator for 14 days.
37 °C
?. [Days -3-0: Pre-differentiation]
Aspirate media from iPSCs and wash with DPBS.
?. [Days -3-0: Pre-differentiation]
Add Accutase and incubate at   for ; if necessary, incubate for additional time up to .
37 °C
?. [Days -3-0: Pre-differentiation]
Use gentle agitation to release cells, and collect in an Eppendorf tube or conical with DPBS.
?. [Days -3-0: Pre-differentiation]
Spin cells at  for  resuspend in N2 pre-diff media.
Centrifuge: 200 34
?. [Days -3-0: Pre-differentiation]
Count cells and add desired amount to an Eppendorf tube or conical, spin, resuspend in N2 pre-differentiation media, and plate onto Matrigel-coated cell culture vessels for the pre-differentiation. Reference seeding density chart for plating
?. [Days -3-0: Pre-differentiation]
Perform ½ pre-differentiation media change every day (w/dox, no RI) throughout days 0-3 pre-differentiation period or full media change on day -1 or day -2. Be consistent.
?. [Days -3-0: Pre-differentiation]
Going into differentiation, it is best if cells are in single-cell suspension. Optional: use cell strainer.
?. [Days -3-0: Pre-differentiation]
Pre-differentiated cells can be frozen in 10%DMSO + N2/B27 Differentiation media (**can also freeze in pre-diff media).
?. [Day 0: Releasing and Plating Pre-Differentiated iNeurons]
Make N2/B27 Differentiation Media:   ABCDEF1ComponentStock ConcentrationFinal ConcentrationDilution FactorFor 100mL (mL)For 50 mL (units in cell)2DMEM/F12*1X0.5X25025mL3Neurobasal-A1X0.5X25025mL4NEAA100X1X1001500uL5GlutaMAX100X0.5X2000.5250uL6N2 Supplement100X0.5X2000.5250uL7B27-VA Supplement50X0.5X1001500uL8NT-310ug/mL10ng/mL10000.150uL9BDNF10ug/mL10ng/mL10000.150uL10Mouse Laminin**1.0mg/mL1ug/mL10000.150uL11Doxycycline***2mg/mL2ug/mL10000.150uL
ABCDEF1ComponentStock ConcentrationFinal ConcentrationDilution FactorFor 100mL (mL)For 50 mL (units in cell)2DMEM/F12*1X0.5X25025mL3Neurobasal-A1X0.5X25025mL4NEAA100X1X1001500uL5GlutaMAX100X0.5X2000.5250uL6N2 Supplement100X0.5X2000.5250uL7B27-VA Supplement50X0.5X1001500uL8NT-310ug/mL10ng/mL10000.150uL9BDNF10ug/mL10ng/mL10000.150uL10Mouse Laminin**1.0mg/mL1ug/mL10000.150uL11Doxycycline***2mg/mL2ug/mL10000.150uL
?. [Day 0: Releasing and Plating Pre-Differentiated iNeurons]
Aspirate media and wash with DPBS.
?. [Day 0: Releasing and Plating Pre-Differentiated iNeurons]
Add Accutase and incubate at  for ; if necessary, incubate for additional time up to.
37 °C
?. [Day 0: Releasing and Plating Pre-Differentiated iNeurons]
Use gentle agitation to release cells, and collect in an Eppendorf tube or conical with DPBS.
?. [Day 0: Releasing and Plating Pre-Differentiated iNeurons]
Spin cells at for .
Centrifuge: 200 34
?. [Day 0: Releasing and Plating Pre-Differentiated iNeurons]
Carefully aspirate DPBS/accutase solution and resuspend in Classic N2/B27 Differentiation Media.
?. [Day 0: Releasing and Plating Pre-Differentiated iNeurons]
Count cells, dilute to appropriate density, and plate onto PDL-coated cell culture vessels.
?. [Day 0: Releasing and Plating Pre-Differentiated iNeurons]
Leftover cells can be frozen down in pre-differentiation media + 10% DMSO.
?. [Day 0+: Neuronal differentiation]
Full media change on day 3 post-plating, once debris is observed.
?. [Day 0+: Neuronal differentiation]
Pipet at the wall of the plate. DO NOT TOUCH BOTTOM OF PLATE! Be extra careful with 96 well plates.
?. [Day 0+: Neuronal differentiation]
Change Classic N2/B27 Differentiation media at minimum once every week without dox or rock inhibitor.
?. [Day 0+: Neuronal differentiation]
Closely monitor. Wait for a week at minimum before using for experiment. Preferably, use after 2 weeks.