Nuclear organization studied with the help of a hypotonic shift: its use permits hydrophilic molecules to enter into living cells.
A new procedure for introduction of hydrophilic molecules into living cells based on efficient uptake of these molecules into the cells during hypotonic treatment is presented and its use is demonstrated by a variety of applications. Experiments with cultured vertebrate and Drosophila cells and various animal tissues demonstrated that the increase in cell membrane permeability under hypotonic conditions is a general phenomenon in all animal cells tested. The efficiency of the method depends on the composition and temperature of the hypotonic buffer, the duration of the hypotonic treatment and the molecular weight of the molecules introduced into living cells. The versatility of this approach is demonstrated with various types of molecules such as modified nucleotides, nucleotides with conjugated fluorochrome, peptides, phosphatase substrates and fluorescent dyes. The method opens new possibilities for the direct investigation of a variety of biological problems as documented here with data on the functional organization of the cell nucleus.
Koberna
K
K
Department of Cell Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Albertov 4, CZ-128 00 Prague 2, Czech Republic.
Stanek
D
D
Malínský
J
J
Eltsov
M
M
Pliss
A
A
Ctrnáctá
V
V
Cermanová
S
S
Raska
I
I
D000546:Amanitins / Q000378:metabolism; D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004396:Coloring Agents / Q000378:metabolism; D004058:Diffusion; D004285:Dogs; D004331:Drosophila melanogaster / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D020869:Gene Expression Profiling; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology*; D007074:Immunoglobulin G / Q000378:metabolism; D007668:Kidney; D008099:Liver / Q000378:metabolism; D008297:Male; D008845:Microinjections; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism; D009924:Organ Culture Techniques; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D051381:Rats; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D014982:Xenopus laevis
D000546:Amanitins; D004396:Coloring Agents; D007038:Hypotonic Solutions; D007074:Immunoglobulin G; D009711:Nucleotides; D004364:Pharmaceutical Preparations; C018009:5-bromouridine triphosphate; D014544:Uridine Triphosphate