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.
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