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TIP
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will end any procedure
Cell Counting
• Prepare dilutions in 1.5 mL microcentrifuge tubes. Other tubes may not be able to accommodate
the width of the sensor, or provide sufficient sample depth for the instrument to function properly.
• Use a diluting solution compatible with the characteristics of cells. The diluting solution used should not
cause changes in the cell size and should have sufficient conductivity to enable operation of the instrument.
Recommended diluting solutions include:
• Dulbecco’s Phosphate buffered saline, D-PBS, with or without calcium and magnesium
(Embryomax
®
D-PBS Cat. No. BSS-1006)
• 10% Fetal Bovine Serum, FBS (Embryomax
®
serum Cat. No. ES-009-B) in D-PBS
• Dulbecco’s Modified Eagle’s Medium, DMEM (Embryomax
®
solution DMEM Cat. No. SLM-220B)
• Complete media (10% FBS in DMEM), and diluted in D-PBS
• 1% dimethyl sulfoxide (DMSO) cell freezing media (Hybri-Max™ DMSO Cat. No. D2650) in D-PBS
• Cell detachment solution (Accutase
®
Cat. No. SCR005)
•
Water, hypotonic, or hypertonic solutions are not acceptable diluting solutions .
Note:
Detergents may interfere with counting; 10% DMSO is not an acceptable diluting solution, but
1% DMSO may be used. Serum-enriched media >10% may also interfere with counting. Dilute samples
with D-PBS, diluted serum in D-PBS (≤10% FBS), DMEM basal medium, DMEM-based complete medium
with ≤10% FBS (undilute or diluted in D-PBS), and Accutase
®
cell detachment solution.
Some suspensions may require a pre-filtration step to remove larger than rated cell clumps, particle populations,
or debris that would clog the sensor’s aperture. A Steriflip
®
filter with 20 µm nylon net is recommend
(use low vacuum
≤
5”Hg).
Cell, Bead, and Particle Suspensions
Materials Required
◊
Cell sample, Scepter™ 3.0 Test Beads (Cat. No. PHCC3BEADS), or particle suspensions
Note:
Some particle suspensions may have a broad diameter range. Debris and/or particles
near or over sensor rating may clog aperture resulting in an
Aperture block error, see p . 20
.
◊
1.5 mL microcentrifuge tubes
◊
Scepter™ 3.0 Sensors
•
The sample volume must be at least 100 μL to draw into the sensor’s microfluidic channels.
• Perform
Installing the Sensor
steps prior to proceeding.
In a 1.5 mL microcentrifuge tube, dilute the single-cell suspension with an appropriate diluting solution
so the cell concentration is within the operating range of the instrument for the sensor being used. The
appropriate dilution will depend on cell type, seeding density, and suspension volume. The volume required
for an accurate count is 100 µL.
Sensor size
Operating Range
Sensor X-Axis
Scale (µm)
Working Diameter
Range (µm)
40 µm
50,000–1,500,000 cells/mL
4-20
5-15
60 µm
10,000–500,000 cells/mL
6-36
8-25
Scepter™ 3 .0 Test Beads
To ensure proper system operation, the Scepter™ 3.0 can be tested periodically with Scepter™ 3.0 Test Beads.
The beads can be used to test the system when first received, as well as for practice and troubleshooting. Refer
to the Scepter™ 3.0 Test Bead container label for expected bead concentration and diameter. The procedure
for testing with Scepter™ 3.0 Test Beads and counting cells is the same, except that the beads are ready to
use and require no dilution. The Scepter™ 3.0 Test Beads are compatible with both 40 and 60 μm sensors.
• Allow Scepter™ 3.0 Test Beads to come to room temperature before use.
• Mix Scepter™ 3.0 Test Beads gently by shaking bead vial for 30 seconds. If using vortex, mix at low speed.
Excessive mixing can lead to inaccurate counts.
