A procedure for selecting and culturing subpopulations of neurons from rat dorsal root ganglia using magnetic beads.
Current protocols for preparing primary sensory neuron cultures are inadequate when studying individual subpopulations of dorsal root ganglion (DRG) neurons. The DRG is made up of a heterogeneous population of cells, making it difficult to study treatment effects on any given population in mass cultures. Thus, we describe a procedure using magnetic beads from Dynal to select and plate viable populations of neurons based on expression of specific cell surface markers. We show that, by the use of the lectin IB4, we can select a highly enriched viable subpopulation of GDNF-responsive DRG neurons, leaving a viable population of non-selected IB4-ve, Trk+ve neurons. Key factors for successful cultures are (i) quick and careful dissection of DRGs from 4- to 5-week-old Sprague-Dawley rats, (ii) adequate removal of debris and non-neuronal contamination and (iii) gentle handling of bead-bound cells during selection.
Tucker
Budd A
BA
Division of Basic Medical Sciences-M5352, Faculty of Medicine, Memorial University of Newfoundland, 300 Prince Philip Dr, St. John's, NL, Canada A1B 3V6.
Rahimtula
Masuma
M
Mearow
Karen M
KM
D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000096:biosynthesis; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000166:cytology* / Q000502:physiology*; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics; D007150:Immunohistochemistry; D037102:Lectins; D008280:Magnetics; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D049329:Nanostructures; D009414:Nerve Growth Factors / Q000378:metabolism; D016900:Neurofilament Proteins / Q000096:biosynthesis; D009474:Neurons / Q000502:physiology*; D009619:Nociceptors / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins
D051100:Glial Cell Line-Derived Neurotrophic Factor; C496198:IB4-saporin conjugate; D007074:Immunoglobulin G; D037102:Lectins; D008565:Membrane Proteins; D009414:Nerve Growth Factors; D016900:Neurofilament Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C051774:neurofilament protein H; D000076985:Saporins; D015740:Calcitonin Gene-Related Peptide