Vi beskriver her, hvordan du udfører en enkelt-celle mikroinjektion af Lucifer Yellow at visualisere cellulær kommunikation via gap-junctions i levende celler, og give nogle nyttige tips. Vi forventer, at dette dokument vil hjælpe alle til at vurdere graden af cellulære kobling skyldes funktionelle gap junctions. Alt her beskrevne kunne være principielt tilpasses til andre fluorescerende farvestoffer med molekylvægt under 1.000 Dalton.
Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.
Gap junctions er intercellulære kanaler, der tillader indbyrdes kommunikation blandt naboceller 1. Denne meddelelse forbinder to eller flere tilstødende celler, hvor hver bidrager med en connexon eller hemichannel at danne den intercellulære kanal. I mammale celler, er det connexon dannet af seks connexiner, monomerer med fire transmembrane domæner og et C og N-terminal i cytoplasmaet 2. Gap junctions ikke kun tillader strømmen af ioner, sekundære budbringere og små metabolitter, men også bidrage til mange former for cellulær kommunikation i mange fysiologiske processer, såsom synaptisk transmission, hjerte sammentrækning, cellevækst og differentiering 3, 4, 5, 6, 7, 8. Desuden gap junctions er blevet forbundet medmange sygdomme, herunder cancer 9, 10, muskelsvind 11, nogle genetiske sygdomme og demyelinisationssygdomme 12.
Denne type af intercellulær krydstale kan evalueres ved adskillige fremgangsmåder 13, 14, 15, 16. I dette papir, viser vi, hvordan du udfører en enkelt-celle mikroinjektion af Lucifer Yellow at visualisere cellulær kommunikation via gap-junctions i levende celler. Vi diskuterer, hvordan man forbereder cellerne og mikropipetten, brugen af mikromanipulator og injektion af Lucifer gult farvestof i en thymus epitelial cellelinje. Normalt kan denne eksperimentelle procedure analyseres ved gennemsnittet af forbundne celler til cellen fyldt med farvestof. Desuden kunne denne fremgangsmåde anvendes med andre fluorescerende farvestoffer med molekylvægt under mellemrummetvejkryds cut-off, som er cirka 1000 dalton.
For at kontrollere tilstedeværelsen af funktionelle intercellulære gap junction, brug af sporstoffer, som er membran uigennemtrængelige, selvom gennemtrængelig af intercellulære kanaler kræves 16. Fluorescein, den første fluorescerende farvestof til at observere celle til celle kobling 22, er gennemtrængelig mellem ikke forbindelsesepitoper membraner 3 og er derfor blevet substitueret med Lucifer gult farvestof 15. I …
The authors have nothing to disclose.
The authors dedicate this paper in honor of Prof. Gilberto Oliveira-Castro who introduced research in intercellular communication by gap junctions in Brazil. This work was funded by Capes, CNPQ and Faperj.
Lucifer yellow | Sigma | L0259 | |
Lithium Chloride | Sigma | L4408 | |
PBS tablets | Sigma | P4417 | |
RPMI | Sigma | R4130 | |
Bovine fetal serum | Cultilab | ||
Trypsin | Sigma | T4799 | |
Microscope | Nikon | TE-2000 | For microinjection experiments, one needs an inverted fluorescence microscope and filters for fluorescent microscopy |
vibration-insulated table | Newport | VH3036W-OPT | A vibration-insulated table is needed to protect the experiments from vibration and avoid cell damage |
Micromanipulator | Narishige | MMO-203 | This equipment allows precision adjustments of the micropipette, which is needed for cell micro injection. |
Current Generator | Digitimer | DS2 | To produce the dye flow through the micropipette, a current below one nano ampere was given using a current generator with an electrode inside the micropipette or an amplifier which has a capacitance compensation circuit (old electrometer) or current injection functions of new patch clamp amplifiers, and the ground wire submersed in the plate dish. Alternatively, the dye can be injected by a pneumatic microinjector, following the factory recommendations. |