This application protocol describes the step-by-step dissociation of PSC-derived cerebral organoids into single cells, using the Neural Tissue Dissociation Kit (P) or (T) in combination with the gentleMACS™ Dissociator. After dissociation, single cells can be either analyzed by flow cytometry using the MACSQuant® Analyzer 10 or can be cultivated for immunostaining and microscopy analysis.
Preparation of the enzyme mixes using Neural Tissue Dissociation Kit (T) or (P)
Enzyme T and Enzyme P of the Neural Tissue Dissociation Kit (NTDK) are ready to use. Prepare aliquots of appropriate volume to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months. Resuspend the lyophilized powder in the vial labeled Enzyme A with 1 mL Buffer A. Do not vortex. This solution should be aliquoted and stored at –20 °C for later use. Avoid repeated freeze-thaw-cycles.
Table 1: Enzyme mixes
|Enzyme mix 1||Enzyme mix 2|
|NTDK (P)||Enzyme P||Buffer X||Buffer Y||Enzyme A|
|50 µL||1900 µL||20 µL||10 µL|
|NTDK (T)||Enzyme T||Buffer X||Buffer Y||Enzyme A|
|200 µL||1750 µL||20 µL||10 µL|
Preparation of stop solution
Prepare a solution of Dulbecco′s Modified Eagle′s Medium (DMEM) containing 20% fetal calf serum.
Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution 1:20 with autoMACS® Rinsing Solution. Keep buffer cold (2−8 °C).
To achieve the appropriate working concentration for safe fixation and permeabilization of cells, the Fixation/Permeabilization Solution 1 (component of FoxP3 Staining Buffer Set) must be diluted 1:4 with the Fixation/Permeabilization Solution 2 (component of FoxP3 Staining Buffer Set) (i.e. for 10⁶ cells use 0.25 mL of Fixation/Permeabilization Solution 1 plus 0.75 mL of Fixation/Permeabilization Solution 2).
To achieve the appropriate working concentration for safe permeabilization of cells, the 10× Permeabilization Buffer (component of FoxP3 Staining Buffer Set) must be diluted 1:10 with deionized or distilled water before use (i.e. 1 mL of 10× Permeabilization Buffer plus 9 mL of deionized/distilled water).
Poly-L-lysine coating of Imaging Plate CG 1.0 (24 well)
Add 500 µL Poly-L-lysine per well and incubate the plate at 37 °C overnight. Discard the Poly-L-lysine directly before plating of cells.
Prepare the culture medium by adding 2% MACS® NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine to the MACS Neuro Medium.
Prepare a solution containing 1% Triton X 100 and 10% fetal calf serum in D-PBS without calcium and magnesium.
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.
autoMACS, CliniMACS, the CliniMACS logo, CliniMACS Prodigy, CryoMACS, CytoMix, CytoStim, DendriMACS, ExiTron, ExpAct, FeraSpin, FeraTrack, GadoSpin, gentleMACS, LIFE 18, LIFE 21, MACS, the MACS logo, MACSductin, MACSelect, MACSfectin, MACSflex, MACSiBead, MACSiMAG, MACSmix, MACSprep, MACSQuant, MACSQuantify, MACSxpress, MidiMACS, MiniMACS, miRXplore, MultiMACS, NeuroBrew, NiraWave, OctoMACS, PepTivator, pMACS, PolySon, PrepProtect, QuadroMACS, REAfinity, REAlease, Rheo, StemMACS, StraightFrom, SuperAmp, SuperMACS, TexMACS, TheraSorb, thermoMACS, TransAct, Tyto, the Tyto logo, VarioMACS, Vio, Viobility, VioBlue, VioBright, VioGreen, Viscover, and µMACS are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. Ficoll-Paque is a trademark of GE Healthcare companies. Vectofusin-1 is a registered trademark of Genethon. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2019 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.