Thermo Fisher Scientific AccuCheck Counting Beads User guide

Brand: Thermo Fisher Scientific

Model: AccuCheck Counting Beads

Type: User guide

AccuCheck Counting Beads

Catalog Number PCB100

Pub. No. MAN0005382 Rev. 2.0

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,

clothing, and gloves. Safety Data Sheets (SDSs) are available from thermoﬁsher.com/support.

Product description

AccuCheck Counting Beads are an ecient single-platform method for absolute cell counts that combines the advantages of direct ﬂow

cytometric immunophenotyping with the use of two dierent ﬂuorescent beads (A and B beads). These two ﬂuorospheres are used as

a double internal standard for blood volume calculation. A known volume of AccuCheck Counting Beads is added to the same known

volume of stained blood in a lyse-no-wash technique. The beads are counted along with cells. Since the concentration of beads is

known, the number of cells per microliter (the absolute count) is obtained by relating the number of cells counted to the total number of

ﬂuorescent bead events. The cell number is then multiplied by the number of total ﬂuorospheres per unit of volume.

Beads A and B have been designed such that their unique characteristics allow the determination of whether they have been

homogenously sampled. Bead A is a 6.4 µm sphere that in ﬂow cytometry presents a low forward scatter (FSC) signal, a lower side

scatter (SSC) signal, and emits broadly when excited with a 488 nm argon laser; however, bead A does not ﬂuoresce when excited with

either a 633 nm helium-neon or 635 nm red diode laser. Bead B is a 6.36 µm sphere that presents a low FSC signal, a slightly higher SSC

signal and a higher ﬂuorescent signal when excited with a 488 nm argon laser. Unlike bead A, bead B ﬂuoresces when excited by a longer

wavelength red laser.

Contents and storage

Product Amount Size Storage

AccuCheck Counting Beads 10 mL 100 tests

• 2–8°C

• Do not freeze

• Protect from light

Procedural guidelines

• This method is not aected by the use of dierent lysing

reagents or varying antibody volumes.

• Relevant cells are deﬁned by their immunoﬂuorescence.

• Use a reverse pipetting technique when pipetting samples

and AccuCheck Counting Beads.

• It is recommended that a threshold or discriminator be set

on the ﬂuorescence channel that includes all the positively

stained cells and the two ﬂuorosphere bead clusters.

• At least 1,000 bead events must be acquired to ensure the

accuracy of the assay.

• It is recommended to use samples that are less than 48

hours old.

• The use of cryopreserved samples is not recommended.

Before you begin

• Bring all reagents to room temperature before use.

• Mix samples gently. Do not vortex.

Prepare the sample

1. Prepare the pipette tip for reverse pipetting using sample.

• For dry tip dispensing, press the pipette plunger to the

second stop in sample, then dispense against the dry

round bottom of the test tube until the ﬁrst pipette stop.

Note: This will leave some residual sample in the pipette tip.

It is recommended that this ﬁrst sample taken for dispensing

be discarded.

• For wet tip dispensing, draw sample to the second

pipette stop. Do two or three slow dispense cycles at

the ﬁrst stop, keeping the pipette tip within the sample.

Dispense at the ﬁrst stop against the lower end of the

wall of the tube.

2. Reverse pipette 100 µl of blood into each tube.

3. To stain cells, add monoclonal antibodies to each tube, mix

gently, then incubate for 10 minutes at room temperature in

the dark.

USER GUIDE

For Research Use Only. Not for use in diagnostic procedures.

4. Add 100 µl of lysing solution to each tube, then vortex.

Note: We recommend using Cal-Lyse™ Lysing Solution (Cat.

No. GAS-010).

5. Incubate for 10 minutes at room temperature in the dark.

6. Add 1 mL of deionized water.

7. Incubate for 5 minutes, then vortex.

8. Mix the AccuCheck Counting Beads by repeated inversion

for 30–45 seconds. Do not vortex.

9. Using the same pipette and reverse pipetting technique

that was used for dispensing the sample, add 100 µL of

AccuCheck Counting Beads to each tube of lysed sample.

10. Cover each sample tube with Paraﬁlm™ ﬁlm.

11. Immediately before analysis by ﬂow cytometry, mix each

sample thoroughly for 30 seconds.

Prepare ﬂow cytometer

Follow manufacturer's instructions to set up the instrument, then

select a protocol that is compatible with the color combination

used.

1. Set a low value for threshold or discriminator in the FSC

channel, then run an unstained and lysed tube.

Note: No additional modiﬁcation of FSC and SSC detectors

is needed to acquire the beads. AccuCheck Counting Beads

can be detected on a FSC vs. SSC dot plot, as well as in the

appropriate ﬂuorescence channel.

2. Draw a region on the FSC vs. SSC dot plot that includes

the leukocyte subpopulations and the two ﬂuorescent bead

clusters.

Acquire data

To ensure accuracy, collect a minimum of 1,000 beads while

collecting an appropriate number of cells.

Analyze data

1. Create a gate selecting all beads (type A and type B) on FSC

vs. SSC dot plot (region R1). Display only region R1 on a

FL2/SSC dot plot.

Figure 1 FSC vs. SSC dot plot

Note: A dierent channel should be used if the staining of

cells interferes with bead analysis.

2. Create a new gate (Region R2) to select all beads, making

sure to exclude debris. Display a FL2/SSC dot plot gated on

R1 and R2.

Figure 2 FL2/SSC dot plot

3. Create two new gates (Regions R3 and R4) to select and

dierentiate type A (lower intensity for FL2) and type B

beads (higher intensity for FL2).

Figure 3 Regions R3 and R4 dot plot

2AccuCheck Counting Beads User Guide

4. Verify on the gate statistics table that the proportion of

each type of gated beads agrees with the manufacturer's

indicated proportions.

5. Record the number of total beads and events in regions of

interest for later calculations.

6. Calculate the absolute number of cell population of interest.

= Absolute Count (cells/μL)

Number of AccuCheck

Counting Beads per μL

Number of cells counted

Total number of beads counted

Thermo Fisher Scientific AccuCheck Counting Beads User guide

Brand: Thermo Fisher Scientific

Model: AccuCheck Counting Beads

Type: User guide

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Thermo Fisher Scientific AccuCheck Counting Beads User guide

Thermo Fisher Scientific AccuCheck Counting Beads User guide

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