Human Dopaminergic Neuron Progenitors. Protocol version 1.2

Transcription

1 Human Dopaminergic Neuron Progenitors Protoco version 1.2

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3 Protoco version 1.2 Tabe of Contents Product Information 2 Recommendations 2 Preparing Dopaminergic Neuron Media 3 Dopaminergic Neuron Differentiation Medium 3 Dopaminergic Neuron Maintenance Medium 3 Coating the Cuture Vesse with SureBond+ReadySet 3 Thawing and Pating Human Dopaminergic Neuron Progenitors 4 Maintenance and Maturation of Human Dopaminergic Neuron Progenitors 5 1

4 Human Dopaminergic Neuron Progenitors Product Information Cataog. No. ax0091 ax0041+ Product Name Format Stock Conc. Human ipsc- Derived Dopaminergic Neuron Progenitors Dopaminergic Neuron Basa Medium 1 miion ces/ via Storage on Arriva Thawing Instructions Storage Once Thawed N/A Liquid Nitrogen Foow protoco N/A 100 ml 1x -20ºC Overnight at 4ºC Suppement A 1 x 160 ml 1x -20ºC Overnight at 4ºC Suppement B 1 x 120 ml 1x -20ºC Overnight at 4ºC SureBond 3 x 120 µl 50x -80ºC Overnight at 4ºC ReadySet 2 x 10 ml 1x 4ºC N/A Store at -20ºC for up to 6 months Store at -20ºC for up to 6 months Store at -20ºC for up to 6 months Store at 4ºC for up to 2 weeks Store at 4ºC for up to 1 month Lot-specific information such as specifications and quaity contro detais are stated in the Certificate of Anaysis. Recommendations Recommended cuture vesse coating: Recommended ce cuture medium: SureBond+ReadySet Differentiation Medium for 5 days foowed by Maintenance Medium Recommended seeding density for assay: 40,000-60,000 viabe ces/cm 2 Recommended centrifugation speed: 400 x g for 5 minutes Recommended days in cuture before assay: 14 days (minimum for tyrosine hydroxyase expression) (5 days in Differentiation Medium then 9 days in Maintenance Medium) 2

5 Preparing Dopaminergic Neuron Media Protoco version 1.2 Dopaminergic Neuron Differentiation Medium Upon receipt aiquot the Dopaminergic Neuron Basa Medium (2 x 20 ml and 4 x 15 ml) and store at or beow -20 C protected from ight. Stored at -20 C, the medium is stabe for 6 months from the date of manufacture. When ready to use, thaw a 20 ml aiquot of Dopaminergic Neuron Basa Medium overnight at 4 C in the dark. On the day of thawing Human ipsc-derived Dopaminergic Neuron Progenitors, transfer 20 ml of aiquoted Basa Medium to a fresh 50 ml tube. Pre-thaw Suppement A on ice and add 80 µl to the 20 ml aiquot of Dopaminergic Neuron Basa Medium. This wi make Differentiation Medium. Store remaining 80 µl of Suppement A at -20 C unti more Differentiation Medium is required. Dopaminergic Neuron Maintenance Medium When ready to use, thaw a 15 ml aiquot of Basa Medium overnight at 4 C in the dark. On day 5 post-seeding, transfer 15 ml of aiquoted Basa Medium to a fresh 50 ml tube. Thaw Suppement B on ice and add 30 μl to the 15 ml aiquot of Basa Medium. This wi make Maintenance Medium. Store the remaining Suppement B (in 30 µl aiquots) at -20 C unti more Maintenance Medium is required. Important! Axo recommends: SureBond+ReadySet coating reagent for Human ipsc-derived Dopaminergic Neuron Progenitors. Coating the Cuture Vesse with SureBond+ReadySet Cacuate the tota surface area that requires coating. Thaw the SureBond coating soution overnight at 4 C. Pre-coat the cuture vesse with ReadySet at a concentration of 250 μl per cm 2. Incubate at 37ºC for 45 minutes. Wash the pate thoroughy four times using an equa voume of sterie distied H 2 O (e.g. if 250 ul of ReadySet is used 250 ul sterie distied H 2 O). During each wash, rock the dish to ensure thorough washing. Do not et the ReadySet dry out foowing washing, proceed straight to coating with SureBond. Diute the SureBond stock soution (50x) in Dubecco s-pbs (1x) (D-PBS, without cacium or magnesium) to make 1x working soution e.g. 120 μl in 6 ml. Coat the surface of the cuture vesse with the SureBond 1x working soution. Axo recommends coating at a voume of 200 μl per cm 2, however, pease optimize for your experiments. Incubate for 1 hour at 37ºC. 3

6 Human Dopaminergic Neuron Progenitors Thawing and Pating Human Dopaminergic Neuron Progenitors On the day before thawing Human Dopaminergic Neuron Progenitors ces, prepare the Differentiation Medium. Pre-warm a media and vesses to 37ºC before use. Aiquot 5 ml of Differentiation Medium into a 15 ml sterie tube and pre-warm at 37ºC. Prepare a second tube with enough Differentiation Medium for ce cuture vesses (see Tabe 1) and pre-warm at 37ºC. Store the remaining medium at 4ºC. To thaw ces transfer the via of ces from iquid nitrogen storage with the via buried in dry ice. Remove the via from dry ice and transfer it immediatey to a 37ºC water bath. Quicky thaw the via of ces by swiring it in a 37ºC water bath. Do not competey submerge the via (ony up to two thirds of the via). Remove the via before the ast bit of ice has meted, after ~1-2 minutes. Do not shake the via during thawing. Take the via of ces to a bioogica safety cabinet, spraying it thoroughy with 70% ethano and wiping with an autocaved paper towe before pacing it in the hood. Once thawed, use a P1000 pipette to transfer the ces drop-wise into a 15 ml sterie conica tube containing 5 ml pre-warmed, 37ºC, Differentiation Medium. Genty wash the via with 1 ml of Differentiation Medium. Transfer this to the 15 ml sterie conica tube containing the ces. Important! Do not mix the ces vigorousy. Avoid generating bubbes. Centrifuge ces at 400 x g for 5 minutes at room temperature Aspirate the supernatant carefuy and resuspend the ce peet with 1 ml of Differentiation Medium. Genty resuspend the ces unti they are in a singe ce suspension. Remove 10 μl of ce suspension and mix it with 10 μl of trypan bue soution. Count the ces. Cacuate the appropriate voume of Differentiation Medium with respect to the cuture vesse (see Tabe 1). Resuspend the ces in Differentiation Medium and pate the resuspended ces at a density ranging from 40,000-60,000 ces/cm 2 on the SureBond+ReadySet coated cuture vesse. Pate the ces drop-wise and eveny on the cuture vesses. Incubate the ces at 37ºC, 5% CO 2. After three days, repace the medium with fresh, pre-warmed, 37ºC, Differentiation Medium. Medium shoud be changed genty in a drop-wise fashion whie pointing the pipette tip toward the wa of the cuture vesse. Refresh the medium every 2 days. Tabe 1: Recommended voumes of medium for different cuture vesses 96-we pate 24-we pate Vesse Type Medium Voume 100 µl/we 500 µl/we 35 mm dish 2 ml 60 mm dish 5 ml 4

7 Maintenance and Maturation of Human Dopaminergic Neuron Progenitors Protoco version 1.2 On day 5 post-seeding, prepare the Maintenance Medium and pre-warm (37ºC) an aiquot. Store the remaining medium at 4ºC. Genty repace the Differentiation Medium with Maintenance Medium. Continue maturation of dopaminergic neurons in Maintenance Medium. Change the medium every 2 days. Dopaminergic neurons can be stained and anaysed 14 days after seeding. After 14 days in cuture, 30% of the dopaminergic neurons shoud be tyrosine hydroxyase positive. Top Tip Dopaminergic neurons shoud be matured for up to 5 weeks after seeding in order to identify dopaminergic neuron receptors and to obtain eectricay active dopaminergic neurons. Longer cuture wi aso increase popuation of astrocytes (GFAP positive). Got any questions? Need hep with the protoco? Contact Axo Technica Support at support@axobio.com Internationa phone US phone AXOL (2965) 5

8 Human Dopaminergic Neuron Progenitors Notes 6

9 Protoco version 1.2 Notes 7

10 Human Dopaminergic Neuron Progenitors Notes 8

11 Notes Protoco version 1.2

12 Address Axo Bioscience Limited Suite 3 The Science Viage Chesterford Research Park Litte Chesterford Cambridgeshire CB10 1XL Emai support@axobio.com Internationa phone US phone AXOL (2965) Web