Apr 07, 2026
iGLUT (NGN2-induced Neuron) Protocol
- Kristen Brennand1,
- Sam Powell2
- 1Yale University School of Medicine;
- 2Icahn School of Medicine at Mount Sinai
- Brennand Laboratory
Protocol Citation: Kristen Brennand, Sam Powell 2026. iGLUT (NGN2-induced Neuron) Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp76bpgzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 07, 2026
Last Modified: May 18, 2026
Protocol Integer ID: 314613
Keywords: iglut neuron, glutamatergic neuron, pure glutamatergic neuron, glutamatergic gene, induced neuron, spontaneous synaptic activity, ngn2, spontaneous synaptic activity by day, iglut
Funders Acknowledgements:
NIMH
Grant ID: R01MH106056
NIMH
Grant ID: R01MH121074
Abstract
Transient overexpression of NGN2 produced iGLUT neurons that were more than 95% pure glutamatergic neurons, robustly expressed glutamatergic genes and produced spontaneous synaptic activity by day 21 in vitro (DIV21).
Materials
**Media Recipes:**
(a) Induction Media
- DMEM/F12+ (already contains sodium pyruvate and glutamax)
- Anti-anti
- N2
- B27
(b) Neuron Media
- BrainPhys
- BDNF
- GDNF
- cAMP
- Ascorbic Acid
- N2
- B27
- Mouse laminin
- Sodium Pyruvate
- Glutamax
- Anti-anti
Harvest iPSCs by incubating in 1mL per well Accutase at 37 degrees C for at 5-10 minutes. Max of 20 minutes
Gently resuspend the iPSCs in the accutase in each well and then mix in a 50mL falcon tube with at least 1:3 volumetric ratio of room temp DMEM.
Spin for 5 minutes (Sam likes 6 b/c he’s paranoid and it makes him feel better) at RT at 800g.
Aspirate supernatant and resuspend pellet in 1 mL of Stemflex with 1:1000 rock inhibitor THX. Count. Dilute in Stemflex with THX to a cell suspension concentration of 1e6 cells/mL.
Add the appropriate amount of Ngn2-Puro and rtTA viruses
A safe amount to add of each is 50 uL per 10e6 cells (and therefore 50uL of each per 10mL) of suspension for Sam’s current aliquots of viruses
Mix well by inversion and gentle, repeated pipetting with a large strip pipet.
Dispense 1.0-1.5 mL of suspension per well (6 well size) coated with 1x matrigel.
Incubate in incubator overnight (min 12 hours; max 36 hours)
DIV1: aspirate the media in the virus hood and replace with Induction media with Dox 1ug/mL
DIV2: replace media with Induction media with dox (1ug/mL) and puromycin (2ug/mL).
DIV3: only if there is a significant amount of cell death, repeat step from DIV2
DIV4: Start AraC 4uM treatment on this day. Continue puromycin 2ug/mL until DIV6. On this day, but no later than DIV6, the cells should be split and replated onto 0.1% PEI and 2x matrigel coated plates OR 4x matrigel coated plates.
(Splitting technique): harvest by incubating in 1mL Accutase at 37degrees for 5-10 minutes. Gently resuspend in the well with the Accutase to break up clumps. Quench with at least 1:3 DMEM. Spin for 5mins at RT at 800g. Resuspend pellet in 1mL of Neuron media with puromycin 2ug/mL and AraC 4uM and THX 1:1000. Count. Dilute to a concentration of 1e6 cells/mL. Mix well by gently by pipetting with large pipet. Dispense 2-4 million cells per well (and therefore 2-4mL of the suspension).
As you can tell, I did NOT include Dox in the media at this step. Dox should be stopped on DIV4 or DIV5….I usually do it when I split on DIV4 or 5.
Next day (DIV5): replace media with neuron media+Puro 2ug/mL+AraC 4uM
DIV6: replace media with just neuron and AraC 4uM.
DIV8: at this time you may stop AraC, so replace media accordingly.
Note: if there is still a concern about flat cells, the AraC can be continued up until DIV10 at the latest. In those cases, I often will decrease it to 2uM AraC at DIV8 and keep until DIV10
DIV10: By now, you should have stopped AraC. For rest of protocol, the cells are just in Neuron Media alone.
Perform ½ media changes 3 times a week or full media changes twice a week until desired time point for harvest
Sam harvest’s his on DIV21.