Analysis of mitochondria isolated from single cells.
Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.
Johnson
Ryan D
RD
Environmental Health Sciences Division, School of Public Health, 140 Warren Hall, University of California Berkeley, Berkeley, CA 94720, USA.
Navratil
Marian
M
Poe
Bobby G
BG
Xiong
Guohua
G
Olson
Karen J
KJ
Ahmadzadeh
Hossein
H
Andreyev
Dmitry
D
Duffy
Ciarán F
CF
Arriaga
Edgar A
EA
D000165:Acridine Orange / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004072:Digitonin / Q000737:chemistry / Q000494:pharmacology; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007834:Lasers; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008928:Mitochondria / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology
D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C413663:fluorescent protein 583; C056258:N(10)-nonylacridine orange; D014357:Trypsin; D000165:Acridine Orange; D004072:Digitonin