Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids

Summary

This protocol reports a unique method of using a streaming cytometer and multiple antibodies for simultaneous assessment of multiple mitochondrial functional parameters, including changes in mitochondrial volume, amounts of the mitochondrial respiratory chain (MRC) complex subunits, and mitochondrial DNA (mtDNA) replication.

Abstract

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain's three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

Introduction

Mitochondria are essential cellular organelles and the major site of the adenosine triphosphate (ATP) production. In addition to providing energy for cells, mitochondria also participate in multiple cellular processes, including cell information transmission, cell differentiation, and apoptosis, and have the ability to regulate cell growth and cell cycle. Changes in mitochondrial function have been identified in various neurodegenerative diseases, including Parkinson's disease (PD)^1,2, Alzheimer's disease (AD)³, and amyotrophic lateral sclerosis (ALS)⁴. M....

Protocol

1. Differentiation of iPSCs into cortical organoids

1. Preparation of matrix-coated plates
   1. Thaw the vial of commercially available basement membrane matrix on ice overnight. Dilute into 1:100 in cold Advanced Dulbecco's Modified Eagle's Medium/Ham's F12 DMEM/F12 (1% final concentration). Make aliquots and store them at -20 °C (see Table of Materials).
   2. Thaw the membrane matrix solution at 4 °C (keep it cold) and coat the required numbe.......

Discussion

A protocol is presented for generating cortical brain organoids from human iPSCs and for performing the flow cytometric analysis of mitochondrial parameters in single cells isolated from these organoids. The cellular composition of the organoids was verified by confocal microscopy with immunohistochemical staining for neuronal and glial cell markers. The flow cytometry-based strategy co-staining with anti-NDUFB10, VDAC 1, and TFAM has been shown to allow the measurement of specific levels of complex I and mtDNA relative .......

Materials

Name	Company	Catalog Number	Comments
Antibodies using in flow cytometry
anti-DUFB10 Alexa Fluor 405	NOVUS biologicals	NBP2-72915AF405
anti-VDAC1 Alexa Fluor 647	Santa cruz technology	sc-390996
anti-TFAM Alexa Fluor 488	Abcam	ab198308
L/D fixable near-IR dead cell stain kit	Life technologies	L10119
Antibodies using in immunofluorence staining
anti-Tuj1	Abcam	ab78078
anti-SOX2	Abcam	ab97959
anti-Alexa Flour 488	Thermo Fisher Scientific	A28175
anti-Alexa Flour 594	Thermo Fisher Scientific	A-21442
Commercial cells
AG05836 (RRID:CVCL_2B58)			Provided by Gareth John Sullivan from the Institute of Basic Medical Sciences at the University of Oslo, Norway
Essential 8 Medium (iPSC culture medium)
Essential 8 Basal Medium	Thermo Fisher Scientific	A1516901
Essential 8 Supplement (50x) 2% (v/v)	Thermo Fisher Scientific	A1517101
Store at 4 °C and warm up to RT before use.
Instruments
Heracell 150i CO2 Incubators	Fisher Scientific, USA
Orbital shakers - SSM1, SSL1	Stuart Equipment, UK
CCD Microscope Camera Leica DFC3000 G	Leica Microsystems, Germany
Water Bath Jb Academy Basic Jba5 JBA5 Grant Instruments	Grant Instruments, USA
Fluid aspiration system BVC control	Vacuubrand, Germany
Leica TCS SP8 STED confocal microscope	Leica Microsystems, Germany
50 mL falcon tube	Sigma-Aldrich	CLS430828
BD LSR Fortessa	BD Biosciences, USA
Flowjo Sampler Analysis	FlowJo LLC, USA
10 mL pipette	Sigma-Aldrich	SIAL1100
1, 10, 100, 1000 mL pipette	Sigma-Aldrich
40 µm Cell stariner	Sigma-Aldrich	CLS431750
ultra-low attachment 96-well plate	S-BIO	MS-9096UZ
Countess II automated cell counter	Thermo Fisher Scientific
Neural differentiation medium  (NDM+)
DMEM/F12	Life technologies	11330032
Neurobasal medium	Life technologies	2110349
Insulin 0.025% (v/v)	Roche	11376497001
MEM-NEAA 0.5% (v/v)	Life technologies	11140050
Glutamax supplement 1% (v/v)	Life technologies	35050
Penicilin/Streptomycin 1% (v/v)	Life technologies	15140-122
N2 supplement 0.5% (v/v)	Life technologies	17502-048
B27 supplement 1% (v/v)	Life technologies	17504-044
β-Mercaptoethanol 50 µM	Sigma-aldrich	M3148
BDNF 20 ng/mL	Peprotech	450-02
Ascorbic acid 200 µM	Sigma-Aldrich	A92902
Store at 4° C for upto 2 weeks
Neural differentiation medium minus viatmin A (NDM-)
DMEM/F12	Life technologies	11330032
Neurobasal medium	Life technologies	2110349
Insulin 0.025% (v/v)	Roche	11376497001
MEM-NEAA 0.5% (v/v)	Life technologies	11140050
Glutamax supplement 1% (v/v)	Life technologies	35050
Penicilin/Streptomycin 1% (v/v)	Life technologies (recheck)	15140-122
N2 supplement 0.5% (v/v)	Life technologies	17502-048
B27 supplement W/O vit. A 1% (v/v)	Life technologies	12587010
β-Mercaptoethanol 50 µM	Sigma-aldrich	M3148
Store at 4° C for upto 8 days
Neural Induction Medium (NIM)
DMEM/F12	Life technologies	11330032
Knockout serum replacement 15% (v/v)	Life technologies	10828028
MEM-NEAA 1% (v/v)	Life technologies	11140050
Glutamax supplement 1% (v/v)	Life technologies	35050
β-Mercaptoethanol 100 µM	Sigma-Aldrich	M3148
LDN-193189 100 nM	Stemgent/Reprocell	04-0074
SB431542 10 µM	Tocris	1614
XAV939 2 µM	Sigma-Aldrich	X3004
Store at 4° C for upto 10 days
Neutralisation medium
IMDM	Life technologies	21980032
FBS 10%	Sigma-Aldrich	12103C
Other reagents
DPBS (Ca2+/Mg2+ free)	Thermo Fisher Scientific	14190250
Bovine Serum Albumin	Europa Bioproducts	EQBAH62-1000
Accutase	Life technologies	A11105-01
Geltrex	Life technologies	A1413302
EDTA	Life technologies	15575038
Advanced DMEM / F12	Life technologies	12634010
Neural tissue dissociation kit	Miltenyi biotec	130-092-628
Y-27632 dihydrochloride Rock Inhibitor	Biotechne Tocris	1254
Fluoromount-G™ Mounting Medium	SouthernBiotech	0100-20
PFA	Thermo Fisher Scientific	28908