文档介绍：流式细胞技术荧光抗体
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Fluorochrome Properties
Desirable properties for fluorochromes:
High
Tend to be very bright—often brighter than the fluorescent protein donor
Disadvantages
High molecular weight (similar to fluorescent protein)
Difficult to make consistently (lot-to-lot variation in emission properties)
Harder to conjugate (same as fluorescent protein)
Some tandems have poor stability
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Nanocrystal Fluorochromes
Advantages
Large Stokes Shift (100−500 nm)
Tend to be very bright
Emission peaks are consistent and narrow, and do not change with variations in the excitation source
Highly resistant to photobleaching
Nanocrystals share biophysical and conjugation properties
Disadvantages
Difficult to conjugate
Instability of bindings
Cytotoxicity
Wide excitation range produces cross-laser spillover
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Excitation and Emission
Excitation wavelengths determine lasers that can excite the fluorochrome.
Emission wavelengths determine filters and PMTs that can measure the emission signal.
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Know Your Cytometer
Cytometer Configuration
BD FACSCantoTM II 4-2-2 configuration is shown below
BDTM LSR II 4-2-2 configuration is similar
4 detectors for blue laser
2 detectors for red laser
2 detectors for violet laser
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Typical Excitation and Emission BD FACSCanto II 4-2-2 (BD LSR II 4-2-2 is similar)
Detector Range
Violet Laser 405 nm
Blue Laser 488 nm
Red Laser 633 nm
410–490 nm
BD Horizon V450 Pacific BlueTM
500–560 nm
BD Horizon V500 AmCyan
515–545 nm
FITC Alexa Fluor® 488
564–606 nm
PE
650–670 nm
APC Alexa Fluor® 647
670–735 nm
PerCP- PE-Cy5 PerCP
750–810 nm
PE-Cy7
BD APC-H7 APC-Cy7
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Fluorochrome Use Depends on the Cytometer Configuration
6-color
8-color
More than 8 colors
FITC, Alexa Fluor® 488