ThermoFisher Scientific invitrogen iSort Series, Manual

Page: 32 / 140

32 iSort
™
Automated Cell Sorter User Guide
Prepare samples
Guidelines for
sample preparation
•
To maximize success, it is essential that samples are prepared in a single-cell
suspension at a density of 10
5
–10
7
cells/mL and are free of aggregates. The
ideal cell concentration is cell type dependent.
•
To prepare single-cell suspensions, use gentle mechanical dissociation, non-
enzymatic methods, or trypsin alternatives (e.g., TrypLE ™ Cell Dissociation
Reagent).
•
Cell clumps and aggregates affect detection and purity and can cause blockages
in the fluidic pathway. Late passaged and contaminated (primary) cell cultures,
and cell cultures that have been allowed to grow beyond confluence are
particularly likely to contain cell clumps and aggregates as a result of DNA and
debris from dead and dying cells.
•
To remove clumps and aggregates, filter the cell sample through cell strainer
before loading it on the instrument.
We recommend using the Corning
™ Falcon ™ Test Tube with Cell Strainer Snap
Cap (35-µm mesh size), available from Fisher Scientific (Cat. No. 08-771-23).
Flowmi
™ Cell Strainers (40-µm mesh size) and Partec CellTrics ™ Filters (30-µm
or 50-µm) also produce single-cell suspensions appropriate for sorting with the
iSort
™
Automated Cell Sorter.
•
If your cells are sticky and filtering does not adequately remove clumps and
aggregates, use EDTA to reduce clumps.
•
Avoid frequent temperature changes to minimize the debris from dead and
dying cells in the sample.
•
Do not run samples containing more than 30% dead cells. We recommend
determining the cell viability of the sample just before running it on the iSort
™
Automated Cell Sorter.
•
The maximum recommended cell sorting rate is 12,000 cells per second, which
also includes the debris detected by the instrument. Sort rates higher than the
recommended maximum can result in lower yield or recovery, and can increase
the likelihood of clogs in the fluidic system.
Note:
Because the flow rate of the iSort
™
Automated Cell Sorter is fixed, the
sorting rate depends solely on the sample concentration.
•
Phosphate buffered saline (PBS) is a common suspension buffer. For best
results, we recommend using flow cytometry grade PBS (Cat. No. A1286301).
•
Place your sample into a 12 × 75-mm sample tube to load it into the instrument
(page 33). The sample tube must contain at least 250 µL of sample volume to
begin sorting or analysis.