Samtidig Optagelse Real-time billeder i Two Emission kanaler ved hjælp af en Dual Camera Emission Opdeling System: Ansøgninger til celleadhæsionsprocesser

Bioengineering

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Carlson, G. E., Martin, E. W., Burdick, M. M. Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion. J. Vis. Exp. (79), e50604, doi:10.3791/50604 (2013).

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Abstract

Multi-color immunofluorescensmikroskopi til påvisning af specifikke molekyler i cellemembranen kan kobles med parallelle plade flow chamber assays til at undersøge mekanismerne for celleadhæsion under dynamiske flow. For eksempel kan cancer-celler mærket med multiple fluoroforer perfunderes i en potentielt reaktiv substrat at modellere mekanismer cancermetastase. Men multi-kanal enkelt kamera systemer og farvekameraer udviser mangler i billedet erhvervelse for real-time levende celle analyse. For at overvinde disse begrænsninger, brugte vi en dobbelt kamera emission splitting til samtidig at indfange real-time billede sekvenser af fluorescens-mærkede celler i strømmen kammeret. Dobbelt kamera emission opdele systemer filter definerede bølgelængde spænder i to monokrom CCD-kameraer, og dermed samtidig erobre to rumligt identiske, men fluoroforen-specifikke billeder. Efterfølgende psuedocolored en kanal billeder kombineres til et enkeltrealtid fusionerede sekvens, der kan afsløre flere målmolekyler på celler bevæger sig hurtigt hen over et område af interesse.

Materials

Name Company Catalog Number Comments
Reagent/Material
BT-20 cells ATCC HTB-19
CHO-E cells Gift from Dr. R. Sackstein (Brigham and Women’s Hospital, Harvard Medical School)
MEM Thermo Scientific SH30024.01
FBS Thermo Scientific SH30396.03
Penicillin-streptomycin Thermo Scientific SV30010
0.25% Trypsin / 0.1% EDTA Thermo Scientific SV30031.01
DPBS Thermo Scientific SH30028.02
DPBS+ Life Technologies 14080-055
BSA Sigma A9647
HECA-452 monoclonal antibody BD Biosciences 555946
Anti-human CD24 monoclonal antibody BD Biosciences 555426
Anti-rat IgM AlexaFluor 488 Invitrogen A21212
Anti-mouse IgG AlexaFluor 568 Invitrogen A11004
Name Company Catalog Number Comments
Equipment
EXi Blue Fluorescence Microscopy Digital CCD Camera Q Imaging EXI-BLU-R-F-M-14-C
Retiga EXi FAST 1394 Digital CCD Camera Q Imaging RET-EXi-F-M-12-C
DC2 Emission Splitter Photometrics DC2
Inverted Fluorescence Microscope Leica DMI6000 B
Streampix 5 software Norpix

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