5um Dual Color Fluorescent Microspheres 0.5%

5 μm Dual-Fluorescent Absolute Counting Beads

SHBC Cyto5UM-10 Double Fluo Microspheres are 5 μm dual-fluorescent beads developed for absolute cell counting by flow cytometry. The microspheres provide both green and red fluorescence signals and support excitation at 488, 535, 561, and 635 nm.

When used with a verified bead number concentration, Cyto5UM-10 serves as an internal counting reference. Researchers can calculate the absolute concentration of target cells by comparing cell events with fluorescent bead events in the same sample.

Key Features

• 5 μm nominal particle diameter

• Green and red dual-fluorescence signals

• Compatible with 488, 535, 561, and 635 nm excitation

• 0.5% solid content

• Easy identification of bead events by fluorescence gating

• Supports single-platform absolute cell counting

• Suitable for conventional and multicolor flow cytometry research

• Liquid microsphere suspension for convenient sample preparation

Product Specifications

For absolute-count calculations, use the lot-specific numerical bead concentration stated in the accompanying product documentation.

How Absolute Counting Beads Work

Cyto5UM-10 microspheres are added to a known volume of cell sample. During flow cytometry acquisition, both target-cell events and fluorescent bead events are collected.

Because the numerical concentration and added volume of the beads are known, the absolute cell concentration can be calculated from the ratio of cell events to bead events. This single-platform approach reduces dependence on a separate hematology analyzer or external cell-counting instrument.

Absolute Cell Count Formula

Absolute cell concentration = (Target cell events ÷ Bead events) × (Bead concentration × Bead volume ÷ Sample volume) × Dilution factor

Where:

• Target cell events are the events within the selected cell gate.

• Bead events are the events within the Cyto5UM-10 bead gate.

• Bead concentration is the lot-specific numerical concentration in beads/μL.

• Bead volume is the exact volume added to the test.

• Sample volume is the original sample volume used in the assay.

• Dilution factor is included when the original sample has been diluted.

The 0.5% solid content describes the mass fraction of microspheres. It does not replace the numerical beads/mL value required for absolute counting.

Recommended Flow Cytometry Workflow

1. Prepare and stain the cell sample according to the validated laboratory protocol.

2. Mix the Cyto5UM-10 suspension thoroughly before pipetting.

3. Add an accurately measured volume of beads to a known volume of sample.

4. Mix gently and acquire the sample on the flow cytometer.

5. Identify the bead population using scatter together with green and/or red fluorescence.

6. Record the target-cell events and bead events.

7. Calculate the absolute cell concentration using the lot-specific bead count value.

8. Apply any sample dilution factor to the final result.

Avoid washing or centrifuging the sample after adding the counting beads, as these steps may cause unequal loss of cells or beads and affect counting accuracy.

Advantages of Dual-Fluorescent Counting Beads

Flexible Bead Identification

Green and red fluorescence signals provide more options for distinguishing Cyto5UM-10 beads from cells, debris, and other particles.

Four Excitation Wavelength Options

Compatibility with 488, 535, 561, and 635 nm excitation supports use with different flow cytometer configurations.

Cell-Relevant Particle Size

The 5 μm particle diameter provides a readily detectable bead population in typical flow cytometry scatter and fluorescence plots.

Single-Platform Absolute Counting

Cells and reference beads are analyzed in the same tube, simplifying the workflow and reducing variation between separate counting platforms.

Suitable for Multicolor Assays

Dual-fluorescence detection allows users to select an appropriate bead gate according to the fluorophores and detectors used in the assay panel.

Applications

Cyto5UM-10 dual-fluorescent counting beads are suitable for research applications including:

• Absolute counting of cultured cells

• Flow cytometric cell concentration measurement

• Immune-cell enumeration research

• Lymphocyte subset analysis

• Stem-cell and cell-therapy process research

• Cell viability assay normalization

• Apoptosis and proliferation studies

• Drug screening and cytotoxicity research

• Whole-blood research workflows after method validation

• Absolute particle counting in validated flow cytometry assays

Instrument and Laser Compatibility

Cyto5UM-10 is designed for flow cytometers equipped with one or more of the following excitation wavelengths:

• 488 nm

• 535 nm

• 561 nm

• 635 nm

The flow cytometer does not necessarily require all four laser lines. Select the excitation and detector combination that provides clear separation between bead events, target cells, and other fluorescent markers.

Actual signal intensity depends on the instrument optics, detector settings, filters, compensation, and assay panel. Users should validate the green and red fluorescence gates on their specific flow cytometer before routine use.

Handling Recommendations

• Store the product according to the conditions stated on the label and product documentation.

• Protect the microsphere suspension from prolonged light exposure.

• Mix thoroughly before each use to obtain a uniform suspension.

• Use calibrated pipettes for both bead and sample volumes.

• Avoid introducing air bubbles or foam during mixing.

• Add beads at the same stage in every test.

• Avoid washing the sample after the counting beads have been added.

• Use consistent acquisition and gating settings when comparing samples.

• Confirm the lot-specific bead number concentration before calculation.

Frequently Asked Questions

What is Cyto5UM-10 used for?

Cyto5UM-10 is used as an internal fluorescent reference for determining absolute cell or particle concentrations by flow cytometry.

What fluorescence signals do the beads provide?

The microspheres provide green and red fluorescence signals, allowing flexible identification of the bead population in different flow cytometry panels.

Which laser wavelengths are compatible with Cyto5UM-10?

The product supports excitation at 488, 535, 561, and 635 nm. Detector and filter settings should be optimized for the individual flow cytometer.

Are all four lasers required?

No. Users can select one or more compatible excitation and detection combinations according to their instrument configuration and assay panel.

Can 0.5% solid content be used directly in the counting formula?

No. Absolute counting requires the numerical bead concentration expressed as beads/mL or beads/μL. Use the lot-specific value provided in the product documentation.

Why use dual-fluorescent beads instead of single-color beads?

Dual green and red fluorescence provides additional gating flexibility and helps distinguish bead events from cells, debris, and assay fluorophores.

Can Cyto5UM-10 be used with any cell type?

It can support many research sample types, but sample preparation, staining, gating, and counting performance must be validated for each cell type and assay.

Is Cyto5UM-10 intended for clinical diagnosis?

Cyto5UM-10 is intended for research use only and is not for use in diagnostic or therapeutic procedures.

Order Cyto5UM-10 from SHBC

Cyto5UM-10 Double Fluo Microspheres are supplied by Shanghai SanYu Biotechnology Co., Ltd. under the SHBC brand.

When requesting a quotation or technical recommendation, please provide:

• Required quantity

• Flow cytometer model

• Available laser wavelengths

• Detector or filter configuration

• Sample type

• Target cell population

• Intended counting range

Contact SHBC for pricing, availability, technical documentation, and flow cytometry application support.