E18.5 mouse DRG dissociation for FACS, 060214 (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Masato Hoshi; Sanjay Jain

Keywords

[‘FACS’, ‘DRG’, ‘RET’, ‘GFP’, ‘nociceptor’]

Subjects

[‘Isolation, purification and separation’, ‘Gene expression analysis’, ‘Experimental organisms’]

Release Date

2017-08-07

Abstract

E18.5 mouse DRG dissociation for FACS, 060214 protocol by the Jain Group

Reagents

• Hibernate-E;LifeTechnologies,#A12476-01
• DMEM/F12;LifeTechnologies,#11330
• 0.05%Trypsin/0.02%EDTA;Sigma,#59417Cor#T3924
• DNaseI;Sigma,#D5025
• Collagenase;Sigma,#C0130
• RNaseinhibitor;RNaseOUT(LifeTechnologies,40units/ul)orequal
• 7-AAD;LifeTechnologies,#A1310(powder,dissolveinDMSOat1mg/ml)

Procedure

See PDF.

1. Euthanize a pregnant mouse following your animal protocol approval and extract embryos from the uterus.
2. Save the embryos/fetuses in a 12-well plate with 3ml of Hibernate-E medium (Life Technologies) in each well of the plate on ice.
3. Take one embryo in 10cm plastic plate with dissection medium (DMEM/F12 (Life Technologies) or Hibernate-E (30ml)) and extirpate DRGs from embryos. (Th11 to S1, total 20 DRGs from each embryo; or as needed for your study) (*when you use DMEM/F12, the medium should be put in a CO2 incubator (cell culture, 5% CO2 incubator at 37C is fine) for more than 2 hours to keep the pH balanced (gauge by the color, prefer orange).
4. Collect DRGs in a 1.5ml tube on ice with dissection medium in a pipette tip. Keep the tube on ice until all dissections are done.
5. Centrifuge at 500 xg for 5min at 4C and discard as much supernatant as possible by using a pipette.
6. Add 500ul (to 1ml, depending on the number of DRGs) of 0.05% Trypsin/ 0.02% EDTA (Sigma) with 200ug/ml of DNaseI (Sigma) to the tube and briefly vortex to mix.
7. Incubate it at 37C for 15min.
8. Triturate with P-1000, then P-200 pipette and dissociate the specimens. Still see some chunks.
9. Add 500ul of of DMEM/F12 + 10% FCS (filtered) and mix well by pipetting.
10. Centrifuge at 500 xg for 5min at 4C and discard supernatant by using a pipette.
11. Add 400ul of DMEM/F12 + 10% FCS (filtered), mix well by pipetting, and then add 600ul of collagenase solution (Sigma, dissolved in DMEM/F12 + 10%FCS (sterilized) at 2mg/ml; final collagenase concentration is 1.2mg/ml). Mix well by pipetting.
12. Incubate it at 37C for 10min.
13. Triturate with P-200 pipette till you notice complete dissociation of the specimen.
14. Centrifuge at 500 xg for 5min at 4C and discard supernatant by using a pipette. Leave around 30ul to avoid disturbing the cell pellet.
15. Add 500ul of PBS + 5% FCS (filtered) with 0.1units/ul of RNase inhibitor (Life Technologies, 40units/ul, 25ul to 10ml PBS/FCS solution) and mix well by pipetting.
16. Centrifuge at 500 xg for 5min at 4C and discard supernatant by using a pipette.
17. Repeat this washing step twice more with PBS/FCS/RNase inhibitor solution. Total three times. In the last wash, the cells get dispersed very quickly with only few rounds of pipetting.
18. After discarding as much supernatant as possible, add 300ul (or appropriate volume for FACS) of PBS + 5% FCS (filtered) with 0.1units/ul of RNase inhibitor and mix well by pipetting.
19. Filter the dissociated cells with 40um pore size cell strainer (BD, #352340) and collect the cells into a FACS tube (BD, #352063).
20. Add 7-AAD at 1ug/ml concentration, if needed, and do sorting. 7-AAD is added 10min before sorting. We have had good success using MoFlo, Sony or AriaII, at 100 um nozzle size, and 30psi.
21. Sorted cells are collected in 750ul of TRIzol-LS (Life Technologies, #10296-010) for RNA extraction and saved in -80C until use.

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium