Cytometry Lab

Example Results

• BFP and GFP analysed on MoFlo.
• Streptavidin-PharRed (Cy7APC) a New Color on the Moflo using the HeNe laser.
• Using UV and 488nm lasers on MoFlo.
• Quantum Dot 655 conjugation.

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BFP and GFP analysed on MoFlo:

• BFP and GFP both from Clontech.
• Both carried into the cells by electroporation using an EFBOS vector.
• Cells were COS.
• Laser1 488nm 180mW.
• Laser3 (Argon) UV 36mW. (In fact, both beams from an Enterprise II)
• FL1 standard "FITC" filter 530DF30, 400V, log.
• FL5 405DF30, 500V, log (we wanted to keep away from the 488nm laser hence the lower-than-optimal pass band of the filter we had on hand).

  Results show good signal/background for both BFP and GFP. The GFP showed very high background staining on cells we believe not to be specifically stained. We are not clear on the reasons for this. Nevertheless, the signal on specifically stained cells was equally very high.

Streptavidin-PharRed (Cy7APC) a New Color on the Moflo using the HeNe laser.

This is a demonstration of mouse spleen cells stained with StreptAvidin-PharRed conjugated to anti-mouse B220-Biotin and Cy5 directly conjugated with anti-mouse CD3.

PharRed (Cy7APC) is a resonance energy transfer dye. It provides a second color for HeNe excitation (633nm) on the Moflo, with an emission at 780nm. The excitation of the first fluorochrome, the donor (APC), is transferred to the other, the acceptor (Cy7), through a nonradiative process (RET).

 

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Using UV and 488nm lasers on MoFlo

Plots show a mixture of blank Calibrite beads (coloured red), FITC Calibrites (coloured green) and Flow Cytometry Standards Hoechst beads (coloured blue). The Hoechst beads obviously fluoresce in the FL1 channel whether or not the UV laser is running. The UV laser does not contribute to the background levels in the FL1 channel but excites some kind of background in all Calibrite beads. No fluorescence compensation has been used. Settings are:

• FL1: 520V, log, 530/40
• FL5: 650V, log, 424DF44

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Quantum Dot 655 Conjugation:

Quantum dots are a newly developed alternative to conventional fluorochrome (see http://probes.invitrogen.com/products/qdot/overview.html for a technical overview).   In general, they excite best at UV wavelengths but their excitation spectra are broad.  A range of QDots is available with discreet emission wavelengths ranging between 525nm and 800nm.  They may be used in flow cytometry (see http://www.nature.com/nm/journal/v12/n8/abs/nm1371.html ) and are also particularly well suited to fluorescence microscopy because of their resistance to photobleaching.   Kits are available for conjugation of QDots to antibody.  The QDot655 kit  (peak emission 655nm) was used to produce these results. QDot655 was conjugated to B220 and compared to a standard PE.B220 conjugate.  PE and UV or violet laser-excited QDot655 fluorescence were analysed on 4 WEHI instruments; Aria. LSR II, DiVa and MoFlo. PE is the brightest conventional fluorochrome used in flow cytometry, however the Qdots performed almost as well as PE on the older WEHI instruments and in fact better than PE on the newer Aria and LSR II instruments. Ref: Catherine Tarlinton (FACS Lab), Kristy O’Donnell (Tarlinton lab).

 

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