RESUMO
Routine quantitative analysis of biomolecule surface density by fluorescence microscopy has been limited by the difficulty of preparing appropriate calibration standards that relate measured fluorescence intensity to actual surface concentration. Supported lipid bilayers are planar fluid films of uniform density and composition which can incorporate a variety of lipidated fluorophores and work well as fluorescence standards. Here, we outline a straightforward strategy to calibrate digital micrographs of fluorescent surfaces such as planar cellular junctions for comparison to supported bilayer standards. It can be implemented with standard microscopy equipment. To illustrate the advantages of this approach, we quantify cell- and bilayer-side protein density patterns in a hybrid immunological synapse between a T-cell and a supported bilayer.
Assuntos
Bicamadas Lipídicas/química , Microscopia de Fluorescência/métodos , Linfócitos T/química , Animais , Calibragem , Fluorometria , Molécula 1 de Adesão Intercelular/química , Molécula 1 de Adesão Intercelular/imunologia , Bicamadas Lipídicas/normas , Camundongos , Fosfolipídeos/química , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologiaRESUMO
The performance of the GROMOS96 parameter set 45A3 developed for aliphatic alkanes is tested on a bilayer of dipalmitoylphosphatidylcholine (DPPC) in water in the liquid-crystalline L(alpha) phase. Variants of the force-field parameter set as well as different sets of simulation conditions or simulation parameter sets are evaluated. In the case of the force-field parameters, the van der Waals constants for the non-bonded interaction of the ester carbonyl carbon and the partial charges and charge group definition of the phosphatidylcholine head group are examined. On the methodological side, different cut-off distances for the non-bonded interactions, use of a reaction-field force due to long-range electrostatic interactions, the frequency of removal of the centre of mass motion and the strength of the coupling of the pressure of the system to the pressure bath are tested. The area per lipid, as a measure of structure, the order parameters of the chain carbons, as a measure of membrane fluidity, and the translational diffusion of the lipids in the plane of the bilayer are calculated and compared with experimental values. An optimal set of simulation parameters for which the GROMOS96 parameter set 45A3 yields a head group area, chain order parameters and a lateral diffusion coefficient in accordance with the experimental data is listed.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Cristalografia/normas , Eletroquímica/métodos , Eletroquímica/normas , Transferência Linear de Energia , Bicamadas Lipídicas/química , Modelos Moleculares , 1,2-Dipalmitoilfosfatidilcolina/normas , Simulação por Computador , Cristalografia/métodos , Campos Eletromagnéticos , Bicamadas Lipídicas/normas , Lipídeos/química , Conformação Molecular/efeitos da radiação , Movimento (Física) , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Eletricidade Estática , Estresse MecânicoRESUMO
Methods of in-vitro artificial formation of bilayer lipid membranes (BLM) and their analytical applications are reviewed, on the basis of 122 literature references. Different techniques for preparation of free-suspended planar BLMs, and gel-, filter-, and solid-supported systems are presented. The analytical applications developed are based on direct interaction of analytes with chemically unmodified BLMs, and with systems modified by use of redox mediators, ionophores, ion-channel forming species, enzymes, antibodies, or DNA.
