Differentiation of dopamine neurons induced by hPSC

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August 28, 2025

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Kit composition information

Name	Specifications	Storage conditions and cycles
Basal Medium 1	220mL	2 ℃-8 ℃, 12 months
Basal Medium 2	210mL	2 ℃-8 ℃, 12 months
Supplement A (50 ×)	4.5mL	-20 ℃, 6 months
Supplement B (10 ×)	1mL	-20 ℃, 6 months
Supplement C (25 ×)	1mL	-20 ℃, 6 months
Supplement D (10 ×)	1mL	-20 ℃, 6 months
Supplement E (50 ×)	4.2mL	-20 ℃, 6 months
Coating solution 1 (250U/mL)	650μL	-20 ℃, 6 months
Coating solution 2 (85.7 U/mL)	1.5mL	-20 ℃, 6 months

Note: If 50 × is the concentration of mother liquor in parentheses, the final concentration should be 1 × when used. For example, supplement A (50 ×) and supplement B (10 ×) need to be added to basal medium 1 to dilute 50 times and 10 times respectively, so that the final concentration of the supplement is 1 ×, and the mNPC induction medium (0-2) is prepared.

A concentration of 250 U/mL of coating solution 1 represents 250 units of coating protein per milliliter, and 2 units of coating protein per square centimeter are needed when plating in this protocol.

Experimental content and method (taking hESC H1 and 1 well of 6-well plate as examples)

1. Reagent preparation

Medium components of each stage

Study Phase	Medium Name	Medium composition of each stage
Midbrain neural progenitor cell stage (mNPC)	mNPC induction medium (0-2)	Basal Medium 1
		Supplement A (1 ×)
		Supplement B (1 ×)
	mNPC induction medium (2-7)	Basal Medium 1
		Supplement A (1 ×)
		Supplement C (1 ×)
	mNPC induction medium (7-9)	Basal Medium 1
		Supplement A (1 ×)
		Supplement D (1 ×)
Midbrain dopamine neuron (mDA) stage	mDA induction medium	Basal Medium 1
		Supplement A (1 ×)
mDA maturation order	mDA maturation medium	Basal Medium 2
		Supplement E (1 ×)

(1) Thaw supplements A, B, C, D, E at 4 ℃, do not thaw at 37 ℃.

(2) In the biological safety cabinet, refer to Table 1 and Table 3, use sterile pipette and gun tip to mix and prepare culture medium for each stage of differentiation according to the proportion. For example, the mNPC induction medium (0-2) consists of basal medium 1 and 1 × concentration of supplements A and B. If the dosage is 10 mL, the preparation method is 8.8 mL basal medium 1 + 200 μL supplement A (50 ×) + 1 mL supplement B (10 ×).

(3) The differentiation medium is recommended to be prepared and used immediately, stored at 4 ℃, and used within 2 weeks.

Note: Supplement B should be protected from light as much as possible when used, and all supplements can be packaged according to the amount used to avoid repeated freezing and thawing.

2. hESC culture and preparation (see the instruction manual of Advance human pluripotent stem cell culture medium for details)

3. DAY 0-9: hESC differentiation into midbrain neural progenitor cells (mNPC)

(1) Thaw the coating solutions 1 and 2 at 4 °C, uniformly mix with DMEM/F12, and spread the plate according to the coating density of 2 U/cm2 for later use.

(2) DAY 0: When the confluence of hESC reaches 75%-85%, aspirate the medium and use 2mL of 1x room temperature PBS (without Ca 2 +/Mg 2 +). The hESC was washed and PBS was aspirated.

(3) Add 1mL of room temperature Accutase containing Y-27632 (10μM), transfer it to a 5% CO2 cell incubator at 37 °C for 3-5min, and allow it to dissociate into single cells.

(4) After 3-5 minutes, the stem cell passage medium was directly added to Accutase in an equal volume, and the cells were gently pipped up and down with P-1000 tips to make a single cell suspension, and the single cell suspension was collected into a 15mL centrifuge tube, and centrifuged at 300 g at room temperature for 5 minutes.

(5) The coating liquid is carefully aspirated from the plate coated with the coating liquid 1 without damaging the coating surface.

(6) At the end of centrifugation, the supernatant was fully removed, and the cells were resuspended with 1 mL of preheated mNPC induction medium (0-2) (components: basal medium 1, 1 × supplement A, 1 × supplement B) (containing 10 μM Y-27632), and gently pipetted up and down to ensure uniform single cell solution.

(7) Cells were counted using an automatic cell counter, dead cells were excluded using trypan blue, cells were seeded onto the coated plate prepared in step 1 so that the final density was 6.0-8.0 × 104 cells/cm2, and the final volume per well was 2 mL (containing 10μM Y-27632) in a 6-well plate, and the well plate was transferred to 37 ℃, 5% CO2 cell incubator for 48 h (if the medium turned yellow after 24 h, the medium was changed (0-2) in time).

(8) DAY 2: Solution exchange using mNPC induction medium (2-7) (components: basal medium 1, 1 × supplement A, 1 × supplement C) until day 7.

(9) DAY 7: mNPC induction medium (7-9) was used to change solution (components: basal medium 1, 1 × supplement A, 1 × supplement D), and culture was performed until day 9. Immunofluorescence staining of cells on day 9 can be used to detect NPC markers such as SOX2, Nestin, and midbrain marker FOXA2.

Note: When using the coating liquid, it should be operated on ice, and all items in direct contact with the coating liquid should be pre-cooled in advance. Try to use the coating solution within 1 week after spreading the plate. Before use, it should be incubated at room temperature for 1h or 37 °C incubator for more than 30 minutes to return to room temperature.

4. DAY 9-23: mNPC induces differentiation into midbrain dopamine neurons (mDA)

(1) DAY 9: On DAY 9, the medium was aspirated from the culture, and the culture was washed with 2 mL of 1 × room temperature PBS (without Ca2+/Mg2 +) per well in a 6-well plate, and then the PBS was removed from the wells.

(2) Add 1mL of room temperature Accutase containing Y-27632 (10μM), transfer it to a 37 °C 5% CO2 cell incubator for 3-5min, and allow it to dissociate into single cells.

(3) After 3-5 minutes, DMEM/F12 (containing 10μM Y-27632) was directly added to Accutase in an equal volume, and the cells were gently pipped up and down with a P-1000 tip to make a single cell suspension. The single cell suspension was collected into a 15mL centrifuge tube and centrifuged at 300 g for 5 minutes at room temperature.

(4) The coating liquid was carefully aspirated from the plate coated with the coating liquid 1 without damaging the coating surface.

(5) After centrifugation, remove the supernatant sufficiently, resuspend the cells with an appropriate amount of preheated mDA induction medium (components: base medium 1, 1 × supplement A) (containing 10 μM Y-27632), and gently pipette up and down to ensure uniform single cell solution. Seed in a plate coated with coating solution 1 at a ratio of 1: 3, add 2 mL of cell suspension to each well and incubate in a 5% CO2 cell incubator at 37 °C for 24 hours.

(6) DAY 11: The mDA induction medium was changed every day or every other day starting from DAY 11 depending on the color of the medium until day 23.

Note: The mDA on D23 day can be frozen in liquid nitrogen with serum-free cell cryopreservation solution plus (abs9478).

5. DAY 23-40: mDA maturity stage

(1) DAY 23: On DAY 23, the medium was aspirated from the culture, and the culture was washed with 2 mL of 1 × room temperature PBS (without Ca2+/Mg2 +) per well in a 6-well plate, and the PBS was aspirated.

(2) Add 1 mL of room temperature Accutase containing Y-27632 (10 μM) to each well, and incubate the culture in a 5% CO2 cell incubator at 37 °C for 5 min.

(3) After 5 minutes, add 1mL DMEM/F12 (containing 10μM Y-27632) to each well, and gently blow the cells to separate from the bottom of the well.

(4) Collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 300 g for 5 min at room temperature.

(5) Carefully aspirate the supernatant without interfering with the cells, resuspend the cells with 1 mL of mDA maturation medium (components: basal medium 2, 1 × supplement E) (containing 10 μM Y-27632) returned to room temperature, and gently pipette up and down to ensure uniform single cell solution.

(6) Trypan blue was used to exclude dead cells, the cells were counted with an automatic cell counter, and the cells were seeded onto a 6-well plate coated with a coating solution 2 prepared in advance so that the density was 6-8 × 10 ^ 5 cells/cm2, and the fluid was changed daily or alternate days depending on the color of the medium until day 25.

(7) DAY 25: Change to mDA maturation medium without Y-27632. The fluid was changed daily or alternate days depending on the color of the medium until day 40.

(8) The expression of neuronal markers (MAP2) and dopamine neuronal markers (TH, DAT, GIRK2) can be detected on day 40.

Note: Due to the high cell density, pay attention to timely fluid change.

Recommended by Xiaoai in this issue

Item number	Product name	Specifications	Storage conditions and cycles
abs90320	hPSC-induced differentiation dopamine neuron kit	1kit
abs9404	Advance human pluripotent stem cell culture medium	100mL	Store at-20 ℃, shelf life is 2 years.
abs812864	Y-27632	10mg	-20 ℃, 1 year (dry powder)
abs9560	DMEM/F12	500mL	Store at 2-8 ℃, protect from light, and the validity period is 12 months.
abs47014935	Cell digestion juice (stem cell grade) (Accutase)	100mL	Store at-20 ℃, shelf life is 2 years.
abs9412	ES/iPS cell cryopreservation solution	100mL	Stored at 2-8 ℃, the shelf life is 12 months.
abs962	PBS	500mL	Stored at 2 ~ 8 ℃ or room temperature, the validity period is 12 months.
abs9478	Serum-free cell cryopreservation solution plus	100mL	Store at 2-8 °C, shelf life 24 months.
abs7033	Cell culture plate (standard clear 6-well plate)	1 box	Room temperature, shelf life 3 years.
abs7034	Cell Culture Plate (Standard Clear 12 Well Plate)	1 box	Room temperature, shelf life 3 years.
abs7035	Cell culture plate (standard clear 24-well plate)	1 box	Room temperature, shelf life 3 years.
abs7119	1.5 mL Centrifuge tube	1 box	Room temperature, shelf life 3 years.
abs7120	15 mL Centrifuge tube	1 box	Room temperature, shelf life 3 years.
abs7121	50 mL Centrifuge tube	1 box	Room temperature, shelf life 3 years.
abs7053	10mL disposable pipette	1 box	Room temperature, shelf life 3 years.
abs7055	50mL disposable pipette	1 box	Room temperature, shelf life 3 years.
abs7072	10uL tips (boxed, sterile and enzyme-free)	1 box	Room temperature, shelf life 3 years.
abs7075	200uL tips (boxed, sterile and enzyme-free)	1 box	Room temperature, shelf life 3 years.
abs7079	1000uL tips (boxed, sterile and enzyme-free)	1 box	Room temperature, shelf life 3 years.

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Absin Bioscience Inc.

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