Download OneComp eBeads

OneComp eBeads
(cat 01-0111-42)
Experimental Procedure
Preparation of Single-Color Compensation Controls
1. Label a tube for each fluorochrome that will be used in the experiment.
2. Mix beads by inverting or pulse-vortexing.
3. Add 1 drop of beads to each tube.
4. Add 1 test of antibody conjugate to each tube.
Note: A test is defined as the amount (μg) of antibody that will stain a cell sample in a final
volume of 100 μL.
5. Mix briefly by flicking or pulse-vortexing.
6. Incubate at 2-8 C
̊ for 15-30 minutes in the dark.
7. Add 2 mL of Flow Cytometry Staining Buffer to each tube and centrifuge at 400-600 x g for
3-5 minutes.
8. Decant supernatant and add 0.2-0.4 mL of Flow Cytometry Staining Buffer to each tube.
9. Mix briefly by flicking before analysis.
Step-By-Step Protocol
•Run unstained cell first to set FSC/SSC and FL PMT voltages
•Briefly run beads to adjust FSC/SSC to visualize beads
•Review all single stained beads to assure they are on scale (PMT voltage should be
decreased if off-scale)
•Run beads- collect data
•Do not use negative beads to set-up voltages.
さらに詳細は次のページの OneComp eBead Protocol Questions をご参照ください。特
に Q4 に関しては非常に大切な情報です。
FAQ
OneComp eBead Protocol Questions
Q1. Is there flexibility in staining incubation times with OneComp eBeads?
Incubations of 5-90 minutes have been tested and may be suitable for most clones. However,
deviation from the suggested 15-30 minute incubation as written in the protocol should be
tested on an individual basis.
Q2. Is it necessary to wash OneComp eBeads after staining?
One wash with 2 mL of Flow Cytometry Staining Buffer (cat. 00-4222) is recommended for
ideal staining of OneComp eBeads. However, it may be possible in some cases to use
unwashed OneComp eBeads, if the antibody staining concentration is low enough. In testing,
it appears that concentrations starting below 0.06 ug per test may provide appropriate
compensation values. Usage in this way should be confirmed in the individual antibody of
interest.
Q3. Is it necessary to use exactly one test of antibody when staining OneComp
eBeads?
No. One test of antibody is recommended for ease of use, since it is the same quantity that
would be added to cells in the experiment. However, since the brightness of the stained
bead is dependent on the bead’s specificity and not on the test antibody’s specificity, it is not
critical that the antibody be used at its optimal concentration. In general, antibodies tested
well between 1.0 ug and 0.03 ug per sample. Some antibodies may need titration for
optimized performance with OneComp eBeads.
Q4. Should fluorescence PMT voltages be set with OneComp eBeads or cells?
Cells. Fluorescence-PMT voltages should be adjusted as desired for unstained cells.
Stained cells and stained beads should then be checked to be sure that the voltage settings
keep the positive populations on scale. If they are off scale, voltages should be adjusted until
the positive populations are on scale. Once you have begun setting up compensation, only
forward scatter and side scatter parameters should be adjusted to properly display beads or
cells. Changes to any other voltages after compensation setup has begun will result in
incorrect compensation settings.
Q5. Can one sample tube of OneComp eBeads be used with multiple antibodies at
once?
No. OneComp eBeads are designed to be used as single-color compensation controls only.
Optimization of OneComp eBeads
Q6. Why do IgM antibodies stain OneComp eBeads less brightly than IgGs and will
this affect my compensation values?
IgM antibodies are pentamers in solution. It is believed that these structures cause some
steric hindrance resulting in less fluorochrome conjugates being captured by the beads.
Compensation values should still be set appropriately using OneComp eBeads.
Q7. My antibody seems to stain the beads dimly. Are there any recommendations on
how to improve the signal?
Rarely, there may be clones that are not captured well by OneComp eBeads. In such cases,
it may be possible to improve the signal brightness by increasing the staining concentration,
the incubation time, or both. In some cases, a different antibody that is conjugated to the
same fluorochrome may be used instead.
Q8. Sometimes my positive population appears as a tight doublet. What should I
define or gate as positive?
There is some antibody-based variation on the shape of the positive peak. When the positive
peak appears as a doublet, it is appropriate to gate on the entire positive population.
Appropriate compensation values will result from this strategy.
Q9. OneComp eBeads are a mixture of negative and positive beads. Can I use a
universal negative in auto-compensation software?
Yes. Since OneComp eBeads are a pre-mixed suspension of both negative and positive
beads, there will always be a tube-specific negative population, but it is not required that the
software use this population. Just include an unstained sample along with your single-color
controls and be sure the gate for positive events is assigned to the positive population for
each sample. The software will then use the universal negative sample and ignore the
tube-specific negative populations. This should be true for any analysis software with
auto-compensation features, including Flowjo and FACSDiva.
General OneComp eBead Questions
Q10. What host species of antibodies do OneComp eBeads work with?
OneComp eBeads capture mouse, rat and hamster (Armenian and Syrian) antibodies of IgG
and IgM classes, independent of light chain. This means OneComp eBeads are
compatible with almost all direct-conjugates used in flow cytometry.
Q11. Do OneComp eBeads work with antibodies of rabbit origin?
While OneComp eBeads were not designed with this intent, they do exhibit some reactivity
to rabbit IgG. In some cases, OneComp eBeads may be useful for compensation of rabbit
antibody conjugates, but this must be determined for each antibody.
Q12. Can I use OneComp eBeads to compensate for Fixable Viability Dyes or Cell
Proliferation Dyes?
No. Since these dyes are not antibodies, they are not compatible with OneComp eBeads.
However, it is possible to use cells for this compensation control while using OneComp
eBeads for the rest of the normal antibody-stain compensation controls.
Q13. Can OneComp eBeads be used with violet laser-excited fluorochromes?
Due to background autofluorescence issues, OneComp eBeads are not optimized for use
with violet laser-excited fluorochromes. However, OneComp eBeads can be used with
eFluor 450- and Pacific Blue-conjugated antibodies because there is no spillover into the
closest detector (FITC) to compensate.
Q14. Are OneComp eBeads compatible with auto-compensation software?
Yes. After staining, OneComp eBeads provide positive and negative peaks that can be used
with auto-compensation software.