 |
This instrument was
procured
to meet the need for sensitive multi-color analysis utilizing the new
digital technologies on a convenient benchtop platform (reducing the
burden on the sorters and core staff). This page describes the
standard layout of the instrument (optics modifications can be made
with advanced notice) as well as control samples necessary to ultilize
the new auto-compensation methodology. |
DiVa 6.0 Software Tutorial (pdf file)
The current configuration is as follows:
488nm Excitation from a Coherent Saphire 20mW solid state laser.
| Detector |
Bandpass Filter |
Fluorochromes* |
| FL1 (D) |
530/30 |
FITC, GFP, Alexa488 |
| FL2 (C) |
575/26 |
PE |
| FL3 (B) |
695/40 |
PerCP-Cy5.5, PE-Cy5.5 |
| FL4 (A) |
780/60 |
PE-Cy7 |
633nm Excitation from a JDS Uniphase 1344P 17mW HeNe laser
| Detector |
Bandpass Filter |
Fluorochromes* |
| FL5 (B) |
660/20 |
APC, Cy5 |
| FL6 (A) |
780/60 |
APC-Cy7 |
408nm Excitation from a Coherent VioFlame 25mW solid state laser
| Detector |
Bandpass Filter |
Fluorochromes* |
| FL7 (B) |
440/40 |
Alexa405, Pacific Blue, Marina
Blue |
| FL8 (A) |
525/50 |
Alexa430, CFP |
351nm Excitation from a Lightwave Xcite 20mW solid state laser
| Detector |
Bandpass
Filter |
Fluorochromes* |
| FL9
(B) |
440/40 |
indo-1
blue |
| FL10
(A) |
530/30 |
Alexa350,
DAPI |
*Many other reagents are utilizable on this machines. To evaluate compatibility, please see BD Reagent Spectrum Viewer (Java App)
Controls Required for Multi-color Flow Acquisition
Compensation Controls: The new digital instrumentation (LSRII and DIVa's) allows for a complete spillover (compensation) matrix to be computed and applied to the experiment. To properly execute this function, the new instrumentation requires the following:
- Unstained cells to determine background for compensation
computation
- Positive controls for each color with the following characteristics:
- A well-defined and bright positive (i.e. CD8,
B220, not
continuums such as CD44)
- Intensity levels at least as bright as your brightest sample (a dimmer control may not properly compensate an extremely bright sample)
- In lieu of the above mentioned criteria, it is possible to use antibody capture beads (Bangs Laboratories Quantum™ Simply Cellular®) to provide a bright positive control utilizing the antibody you will be using in your experiment
Background (FMOC) Controls: In multi-color flow cytometry, the absolute background in a given channel is a function of all the channels that spillover and require compensation in the channel of interest. Extremely bright fluorochromes can add to the level of background in a corrected channel that will limit the sensitivity in that channel. The proper way to evaluate the background is to have a control that is all the other stains that you will be using, except for the color detected in that channel, hence the name "fluorescence minus one control" (FMOC). This control is a must if you are trying to evaluate very dim populations in multi-color space.
Isotype Controls: As always for flow cytometry, it is important to evauate the specificity of your staining and determine whether isotype specific background binding is a problematic factor. With this in mind, it is important to realize that isotype controls are not the proper way to determine what the boundary for positive and negative expression of a marker in multi-color experiments.