GeminiBio MXG102, Руководство пользователя

Страница: 62 / 93

Moxi GO II
™
User Guide
Page 57
Compensation (of Spillover)
Overview/Background
There is an expected spectral overlap when using PMT filter detection in 525/45nm and
561nm/LP for particular fluorophores. Depending on the fluorophores used in a test, and their
relative intensities, the fluorescence signal of a given fluorophore might “spillover” from its
primary fluorescent channel/PMT into the secondary fluorescent channel/PMT. In some cases,
the spillover can interfere with the interpretation of the data. The result of this expected
“spillover” is the data of a single color control runs in a diagonal at increasing MFIs in another
PMT channel (See Below). In those cases, it may be necessary to “compensate” the data to
remove that spillover effect. Compensation is performed, as is traditional, by subtracting a
percentage of the primary flow signal magnitude for a given event from the corresponding value
on the secondary channel. Due to the nature of the filter selection and fluorophore emission
properties, in almost all instances on the Moxi GO II, the spillover will be from the Green
(525/45nm) channel into the Red (561nm/LP) channel. The 646nm/LP filter selection for the red
(PMT2) channel would be expected to minimize this spillover as its edge is set past the
emission tail of most green dyes. However, it is still in a range that can pick up propidium iodide
(PI, viability dye) emission. In this regard, it is ideal for use when using a PI counterstain.
However, the 646nm/LP filter is pushed too far out for Phycoerythrin (PE) detection. So, when
using PE in dual-marker immunolabeling, the 561nm/LP filter must be used.
Moxi GO II
TM
Compensation
General Workflow
To compensate spillover for any experiment, it is first necessary to create single-stain controls
for any fluorophore that needs spillover compensation.
Compensation needs to performed
using a SINGLE-STAIN positive control. It is important not to use a dual-stained sample
as we need certainty that any signal on the spillover channel is a result of spillover, not a
fluorophore that is intended for that channel.
As mentioned above, by nature of the
emission profiles of fluorophores, almost all spillover on the Moxi GO II is from the Green
Channel (525/45nm) into the orange/red (561nm/LP) channel. However, there may be cases
when an Orange/Red dye backspills. In those cases, a single stain control would need to be
prepared for each dye. The general workflow would then be:
1. Run a negative control (unlabeled) sample (if you expect the single-stain control
sample will be 100% positive/fluorescent). These samples will follow the “Load
Control Set” compensation approach described in the section below.
2. Run each single stain control on the Moxi GO II using the same assay that you
intend to use for the overall experiment.
3. PRIOR TO SAVING the single-stain control, adjust the compensation using the
approaches described below.
4. Save the data.