5um Double Fluo Microspheres 0.5%

Flow Cytometry Absolute Counting Fluorescent Microspheres

SHBC Flow Cytometry Absolute Counting Fluorescent Microspheres are dual-fluorescent reference particles developed for absolute cell enumeration, flow cytometric immunophenotyping, immune-cell analysis and quantitative particle-counting research.

The Double-Fluo Microspheres feature two fluorescence detection channels:

• Excitation at approximately 488 nm with emission centered near 530 nm

• Excitation at approximately 633 nm with emission centered near 670 nm

The microspheres are supplied as an aqueous suspension with a solids content of 0.5%.

The dual-laser, dual-emission fluorescence profile allows the microspheres to be identified using both blue-laser and red-laser channels on compatible flow cytometers. When a known volume of microspheres with a lot-specific, assayed particle concentration is added to a known sample volume, the absolute concentration of cells or other particles can be calculated from the ratio of sample events to microsphere events.

This product is intended for research institutions, flow cytometry laboratories, immunophenotyping reagent developers, antibody companies and manufacturers requiring development samples, pilot quantities or bulk fluorescent microsphere supply.

Product Overview

Flow cytometry commonly reports the percentage of cells within a selected population. However, many research applications also require the actual number of cells per microliter, milliliter or original sample volume.

Absolute counting microspheres provide an internal particle reference for this calculation.

A known volume of counting microspheres is added directly to the cell sample. The flow cytometer then records the target-cell events and microsphere events during the same acquisition.

Because the microsphere concentration is known, the number of cells in the original sample can be calculated from the ratio between the two event populations.

Double-Fluo Microspheres provide two fluorescence responses for microsphere identification:

• Green fluorescence near 530 nm under approximately 488 nm excitation

• Far-red fluorescence near 670 nm under approximately 633 nm excitation

The dual-fluorescent profile can help users:

• Distinguish counting microspheres from unstained cells

• Separate microspheres from cellular debris

• Confirm microsphere identity in two fluorescence channels

• Reduce dependence on scatter parameters alone

• Establish a compact and reproducible microsphere gate

• Support flow cytometers equipped with blue and red lasers

• Develop customized absolute-counting reagents

• Improve internal acquisition and process control

The microspheres may be evaluated with whole blood, lysed whole blood, peripheral blood mononuclear cells, cultured cells, isolated immune cells and other particle suspensions.

Compatibility must be verified with the intended sample matrix, antibody panel, cytometer and data-analysis workflow.

Key Features of Double-Fluo Microspheres

Dual Excitation at 488 nm and 633 nm

The Double-Fluo Microspheres are designed for excitation using two common flow cytometer laser lines:

• Approximately 488 nm blue-laser excitation

• Approximately 633 nm red-laser excitation

The 488 nm excitation provides a green fluorescent response that can be detected near 530 nm.

The 633 nm excitation provides a far-red fluorescent response that can be detected near 670 nm.

This dual-laser design supports flow cytometers equipped with both blue and red laser modules and allows reagent developers to select one or both fluorescence signals for microsphere identification.

Instruments using 635 nm or 640 nm red lasers may also be evaluated. Final compatibility depends on laser power, filter configuration, detector sensitivity and the lot-specific fluorescence spectrum.

Dual Emission at 530 nm and 670 nm

The nominal fluorescence emission channels are:

• Approximately 530 nm after 488 nm excitation

• Approximately 670 nm after 633 nm excitation

The 530 nm signal is generally located in the green fluorescence detection region.

The 670 nm signal is located in the far-red fluorescence detection region.

The two fluorescence signals may be used individually or together according to the antibody panel and cytometer configuration.

A dual-fluorescence gate can provide an additional method for distinguishing counting microspheres from:

• Unstained cells

• Cell fragments

• Platelets

• Electronic noise

• Autofluorescent events

• Single-channel fluorescent particles

• Nonspecific sample contaminants

The exact filter center wavelengths and bandwidths must be verified on the intended flow cytometer.

0.5% Solids Aqueous Suspension

The product is supplied with a solids content of 0.5%.

When expressed as weight per volume, 0.5% solids corresponds to approximately 5 mg of microspheres per milliliter.

The solids content is useful for:

• Manufacturing process control

• Formulation development

• Dilution calculations

• Batch-to-batch mass comparison

• Customized concentration preparation

• Filling-process control

However, solids content is not the same as the particle number concentration required for absolute counting.

Lot-Specific Bead Concentration

Reliable absolute counting requires a lot-specific, assayed particle number concentration stated as:

• Beads/mL

• Beads/μL

• Beads per recommended test volume

The bead number concentration must be measured using a validated particle-counting method and documented on the product label or certificate of analysis.

This value is used in the absolute cell-count calculation.

Important controls include:

• Particle number concentration

• Concentration tolerance

• Filling accuracy

• Bottle-to-bottle consistency

• Suspension homogeneity

• Sampling reproducibility

• Stability during storage

• Stability after transportation

Commercial absolute-counting products similarly rely on calibrated or lot-specific microsphere concentrations rather than solids content alone.

Uniform Particle Size

Controlled particle size helps generate a compact microsphere population on forward-scatter and side-scatter plots.

A narrow particle-size distribution can improve:

• Gating reproducibility

• Event identification

• Instrument-to-instrument transfer

• Sampling consistency

• Doublet discrimination

• Batch-to-batch comparison

• Automated data analysis

The nominal particle size and permitted size range should be stated in the final product specification.

Uniform Dual-Fluorescence Intensity

Consistent fluorescence intensity in both channels helps produce a clearly separated microsphere gate.

Recommended fluorescence quality attributes include:

• Mean green fluorescence intensity

• Green fluorescence coefficient of variation

• Mean far-red fluorescence intensity

• Far-red fluorescence coefficient of variation

• Percentage of dual-positive microspheres

• Ratio of green to far-red fluorescence

• Fluorescence stability during storage

• Instrument-to-instrument detectability

The two fluorescence signals should be sufficiently separated from the intended cell populations and antibody conjugates.

Internal Fluorescent Labeling

The fluorescent dyes are incorporated into the microsphere structure rather than relying only on weak surface adsorption.

Internal fluorescent labeling can help:

• Reduce dye loss

• Improve washing resistance

• Maintain a stable outer surface

• Protect the fluorescent material

• Improve storage stability

• Support long-term reagent development

• Reduce lot-to-lot fluorescence variation

The finished product should still be protected from prolonged exposure to strong light.

Single-Platform Absolute Counting

The microspheres and target cells are acquired during the same flow cytometry run.

This allows the user to calculate cell concentration without relying exclusively on a separate hematology analyzer or external particle counter.

The single-platform method can reduce variation introduced by transferring percentage results between different instruments. Official counting-bead products from Thermo Fisher and Beckman Coulter use the same ratio-based principle.

Suitable for Reagent Development and Bulk Manufacturing

SHBC Double-Fluo Microspheres are positioned for customers requiring more than a small research package.

Available project stages may include:

• Initial microsphere evaluation

• Flow cytometer compatibility testing

• Gating-method development

• Absolute-count reagent development

• Antibody-panel compatibility testing

• Pilot manufacturing

• Stability studies

• Process validation

• Bulk production

• Lot reservation

• OEM and ODM manufacturing

Technical Specifications

The following information should be confirmed and added before formal publication:

• Catalog number

• Nominal particle size

• Particle-size tolerance

• Microsphere matrix

• Lot-specific beads/mL

• Particle-count concentration tolerance

• Suspension buffer

• Surfactant

• Preservative

• Package size

• Storage temperature

• Shelf life

Solids Content vs. Bead Number Concentration

Solids content and particle number concentration describe different product properties.

What Does 0.5% Solids Mean?

A 0.5% weight-per-volume suspension contains approximately:

5 mg of microspheres per milliliter

Solids content describes the total mass of microsphere material in a given volume.

It is useful for manufacturing, dilution and formulation control.

What Does Beads/mL Mean?

Beads/mL describes the number of individual microspheres in one milliliter.

This value depends on:

• Microsphere diameter

• Particle density

• Particle-size distribution

• Solids concentration

• Aggregate level

• Particle-counting method

Two microsphere products can have the same 0.5% solids content but very different beads/mL values if their particle sizes are different.

Which Value Is Used for Absolute Counting?

The absolute cell-count formula requires the calibrated particle number concentration, not only the solids content.

The COA should therefore provide a lot-specific concentration such as:

• 8.5 × 10⁵ beads/mL

• 1.0 × 10⁶ beads/mL

• A specified number of beads per recommended test volume

The examples above illustrate reporting formats only and are not product specifications.

How Absolute Counting Microspheres Work

A known volume of Double-Fluo Microspheres is added to a known volume of the sample.

During acquisition, the flow cytometer records:

• The number of target-cell events

• The number of counting-microsphere events

Because the number of microspheres added to the tube is known, the original concentration of target cells can be calculated from the event ratio.

A typical process includes:

1. Prepare or stain the cell sample.

2. Lyse red blood cells if required.

3. Mix the counting microsphere suspension thoroughly.

4. Add an accurately measured volume of microspheres.

5. Acquire the sample without losing cells or microspheres.

6. Gate the target-cell population.

7. Gate the Double-Fluo Microsphere population.

8. Record cell and microsphere event numbers.

9. Calculate the absolute cell concentration.

10. Apply any required sample-dilution factor.

Absolute Cell Count Calculation Formula

The general calculation is:

Absolute cell concentration = Cell events ÷ Bead events × Bead concentration × Bead volume ÷ Sample volume × Dilution factor

When the result is expressed as cells per microliter:

Cells/μL = (Cell events / Bead events) × Bead concentration in beads/μL × (Bead volume / Sample volume) × Dilution factor

Where:

• Cell events are events within the validated target-cell gate.

• Bead events are events within the validated Double-Fluo Microsphere gate.

• Bead concentration is the lot-specific value stated on the COA.

• Bead volume is the actual volume of bead suspension added.

• Sample volume is the original sample volume used for the calculation.

• Dilution factor accounts for any applicable dilution.

Simplified Calculation

When the bead and sample volumes are equal:

Cells/μL = Cell events ÷ Bead events × Bead concentration in beads/μL

This simplified formula is only valid when the sample-preparation design supports the assumption.

Thermo Fisher and Beckman Coulter counting-bead methods use the same underlying ratio between target-cell events and assayed microsphere events.

Why Use Double-Fluo Counting Microspheres?

Confirm Microsphere Identity in Two Channels

A counting population identified using only scatter or one fluorescent signal may overlap with sample debris or other fluorescent events.

A dual-fluorescent signature provides an additional level of microsphere identification.

Potential gating options include:

• 530 nm fluorescence alone

• 670 nm fluorescence alone

• 530 nm versus 670 nm

• FSC versus 530 nm

• SSC versus 670 nm

• Dual-positive microsphere gating

Reduce Dependence on Scatter Alone

Forward and side scatter can vary with:

• Cytometer model

• Laser alignment

• Detector voltage

• Sample matrix

• Particle refractive index

• Threshold settings

• Instrument maintenance

Fluorescence-based identification can help maintain microsphere detection when scatter separation is limited.

Support Blue- and Red-Laser Instruments

Many multicolor flow cytometers contain both blue and red lasers.

Double-Fluo Microspheres allow manufacturers to develop counting reagents compatible with:

• Blue-laser detection

• Red-laser detection

• Dual-laser confirmation

• Different filter configurations

• Different antibody panels

Improve Internal Quality Control

When a defined dual-fluorescence relationship is established, users can monitor:

• Loss of one fluorescence signal

• Incorrect gate placement

• Optical-channel failure

• Sample contamination

• Microsphere aggregation

• Abnormal signal ratio

• Instrument-setting changes

Support Automated Analysis

A compact, dual-positive microsphere population can help simplify automated identification by analysis software.

Automated gating should still be validated across:

• Multiple lots

• Multiple instruments

• Multiple operators

• Different sample matrices

• Low and high cell concentrations

Flow Cytometer and Detector Compatibility

Blue-Laser Channel

The nominal blue-laser configuration is:

• Excitation: approximately 488 nm

• Emission: approximately 530 nm

Possible initial detector configurations include filters centered near the green fluorescence region.

The actual filter bandwidth must be selected according to the microsphere spectrum and the antibody panel.

Red-Laser Channel

The nominal red-laser configuration is:

• Excitation: approximately 633 nm

• Emission: approximately 670 nm

Instruments using 635 nm or 640 nm red lasers may be compatible after verification.

Antibody Panel Considerations

The 530 nm channel may overlap with common green-emitting antibody conjugates.

The 670 nm channel may overlap with far-red fluorophores.

During panel development, evaluate:

• Fluorescence overlap

• Spectral spillover

• Compensation requirements

• Detector saturation

• Microsphere brightness

• Cell-marker intensity

• Available unused channels

The microsphere gate may be established using the channel that provides the best separation from the stained cell populations.

Conventional and Spectral Cytometers

Double-Fluo Microspheres may be evaluated on:

• Conventional filter-based flow cytometers

• Spectral flow cytometers

• Benchtop flow cytometers

• Automated flow cytometry analyzers

• Cell-analysis platforms with volumetric sampling

• Research cell sorters

For spectral cytometers, the full fluorescence signature should be measured and included in the reference library if appropriate.

Recommended Applications

Absolute Immune-Cell Enumeration

The microspheres may be evaluated for absolute enumeration of:

• Total leukocytes

• T cells

• CD4-positive T cells

• CD8-positive T cells

• B cells

• Natural killer cells

• Monocytes

• Granulocytes

• Dendritic-cell populations

• Other gated immune-cell subsets

The target population is defined using the laboratory’s validated antibody panel and gating strategy.

Flow Cytometric Immunophenotyping

Double-Fluo Microspheres can be added to immunophenotyping workflows to convert cell percentages into absolute numbers.

Potential research formats include:

• Whole-blood immunophenotyping

• Lyse-no-wash assays

• PBMC analysis

• Bone-marrow research

• Cell-culture analysis

• Immune-monitoring studies

• Preclinical research

• Cell-therapy process development

Cell Culture and Bioprocess Monitoring

Potential research applications include:

• Cultured immune-cell counting

• T-cell expansion monitoring

• NK-cell culture monitoring

• Cell-recovery studies

• Cell-manufacturing process development

• Cell viability and yield research

• Bioprocess sampling

Viability dyes and counting microspheres can be combined when the fluorescence panel provides sufficient channel separation.

Stem and Progenitor Cell Research

The microspheres may be evaluated in research workflows involving:

• CD34-positive cell enumeration

• Hematopoietic progenitor research

• Cell-processing recovery

• Cryopreservation studies

• Cell-therapy process development

Method-specific validation is required.

Microorganism and Particle Counting

After appropriate validation, the same ratio-based principle may be evaluated for:

• Yeast

• Bacteria

• Microalgae

• Synthetic particles

• Extracellular vesicle preparations

• Environmental particles

Particle size, scatter threshold and fluorescence separation must be suitable for the target.

Instrument and Method Development

The product may also support:

• Flow cytometer method development

• Sample-preparation optimization

• Pipetting-method validation

• Operator training

• Software-gating development

• Counting-precision studies

• Inter-instrument comparison

• OEM counting reagent development

Recommended Flow Cytometry Workflow

Step 1: Prepare the Sample

Prepare a known volume of the biological sample.

Record:

• Original sample volume

• Any predilution

• Staining volume

• Lysis volume

• Final dilution factor

• Any washing step

Loss of cells during washing can affect the relationship between the measured result and the original sample.

Step 2: Stain the Cells

Add the required antibody panel and incubate according to the validated method.

Protect fluorescent reagents from unnecessary light exposure.

Step 3: Lyse Red Blood Cells if Required

For whole-blood applications, use a validated lysis method.

A lyse-no-wash workflow can reduce cell loss, but compatibility with the microsphere suspension and antibody panel must be confirmed.

Step 4: Mix the Microspheres

Immediately before pipetting:

• Bring the product to the required operating temperature.

• Mix according to the validated instructions.

• Confirm that no settled layer remains.

• Avoid generating foam.

• Avoid prolonged high-speed vortexing unless validated.

• Inspect for visible aggregates.

AccuCheck recommends careful mixing and reverse pipetting to improve counting accuracy.

Step 5: Pipette an Accurate Microsphere Volume

Use a calibrated pipette.

For critical quantitative work, consider:

• Reverse pipetting

• Low-retention tips

• Consistent aspiration depth

• Consistent pipetting speed

• Wet-tip dispensing

• Gravimetric pipette verification

• Replicate dispensing studies

Step 6: Acquire the Sample Promptly

After adding the microspheres:

• Mix gently.

• Avoid prolonged delay before acquisition.

• Keep the sample protected from light.

• Avoid unnecessary settling.

• Maintain consistent acquisition timing.

Step 7: Collect Sufficient Microsphere Events

For development work, collecting at least 1,000 microsphere events is a useful starting target, based on the AccuCheck workflow. The final event requirement should be established from the precision needed for the specific method.

Low microsphere event numbers can increase statistical uncertainty.

Step 8: Apply the Validated Gates

Record:

• Total microsphere events

• Target-cell events

• Excluded aggregate events

• Acquisition time

• Total analyzed volume, when available

• Instrument settings

Step 9: Calculate the Result

Use the lot-specific microsphere concentration and the validated formula.

Do not substitute the 0.5% solids content for the particle number concentration.

Suggested Gating Strategy

Gate 1: Time Stability

Plot time against one fluorescence channel or total event rate.

Exclude periods showing:

• Interrupted flow

• Sample clogs

• Sudden event-rate changes

• Air bubbles

• Microsphere settling

• Sample aspiration instability

Gate 2: Broad Particle Gate

Use FSC and SSC to include:

• Target cells

• Counting microspheres

Exclude:

• Electronic noise

• Very small debris

• Extreme aggregates

Do not set the threshold so high that counting microspheres are removed.

Gate 3: Microsphere Fluorescence Gate

Plot 530 nm fluorescence against 670 nm fluorescence.

Define the validated Double-Fluo Microsphere region.

The microsphere population should show a consistent relationship between the two fluorescence signals.

Gate 4: Singlet Microspheres

Where pulse geometry is available, use:

• Area versus height

• Area versus width

Exclude microsphere aggregates and coincident events.

Gate 5: Target-Cell Population

Apply the laboratory’s validated immunophenotyping gates.

Potential markers may include:

• CD45

• CD3

• CD4

• CD8

• CD19

• CD16

• CD56

• CD14

• Viability markers

Gate 6: Final Event Review

Confirm:

• Microsphere gate separation

• Adequate bead event count

• Stable bead fluorescence ratio

• Expected cell-population distribution

• Absence of major acquisition instability

• Valid controls

Factors Affecting Absolute Counting Accuracy

Microsphere Concentration Accuracy

The beads/mL value directly affects the calculated cell result.

An error in the assigned microsphere concentration produces a proportional error in the calculated cell concentration.

Pipetting Accuracy

The calculation assumes that the stated volume of microspheres was actually added.

Potential sources of error include:

• Pipette calibration

• Tip retention

• Incomplete dispensing

• Air bubbles

• Incorrect aspiration depth

• Microsphere settling

• Operator technique

Thermo Fisher notes that counting accuracy depends strongly on sample handling and precise bead-volume delivery.

Suspension Homogeneity

Microspheres may settle over time.

If the suspension is not homogeneous, different aliquots may contain different numbers of microspheres.

Aggregate Formation

Aggregates may be counted as:

• One event

• An excluded event

• A cell-like event

• An abnormal fluorescent event

Aggregate control is therefore important for accurate counting.

Event Statistics

The precision of a ratio-based calculation depends on the number of recorded cell and microsphere events.

Very low event counts increase statistical variation.

Gating Consistency

Changing the microsphere or cell gate between samples can change the calculated result.

Automated or locked analysis templates may improve consistency after validation.

Threshold Settings

A high FSC or SSC threshold can remove small counting microspheres.

A fluorescence-based threshold may be considered when compatible with the full antibody panel.

Sample Loss

Centrifugation, aspiration and washing can remove cells or microspheres.

The calculated result must be interpreted according to the sample-preparation workflow.

Coincidence and High Event Rate

Excessive event rates can cause coincident passage of multiple particles through the detection point.

Dilute the sample or reduce flow rate when required.

Instrument Stability

Changes in:

• Laser power

• Fluidics

• Optical alignment

• Detector voltage

• Filter performance

• Flow rate

can affect microsphere identification and counting reproducibility.

Troubleshooting Guide

Microspheres Are Not Visible

Possible causes:

• Threshold is too high

• Incorrect fluorescence channel

• Incorrect laser selected

• Detector voltage is too low

• Microspheres have settled

• Sample was not mixed

• Fluorescence filter is incompatible

• Microsphere concentration is too low

• The product was exposed to damaging conditions

Recommended actions:

1. Reduce the scatter threshold.

2. Check the 530 nm and 670 nm channels.

3. Confirm laser availability.

4. Mix the suspension thoroughly.

5. Run microspheres without cells.

6. Compare with the lot-specific reference data.

Microsphere Gate Is Too Broad

Possible causes:

• Aggregation

• Inadequate mixing

• Excessive vortexing

• Contamination

• Detector saturation

• Mixed singlet and doublet events

• Instrument instability

Recommended actions:

• Apply singlet gating.

• Reduce detector gain if saturated.

• Inspect the suspension.

• Use clean tubes and filtered buffers.

• Compare the result with a retained reference lot.

Green Signal Is Strong but Far-Red Signal Is Weak

Possible causes:

• Red laser is unavailable or incorrectly configured

• Far-red detector voltage is too low

• Filter center wavelength is unsuitable

• Red-laser alignment is poor

• Fluorescence has changed during storage

• Incorrect analysis channel was selected

Far-Red Signal Is Strong but Green Signal Is Weak

Possible causes:

• Blue laser is unavailable or incorrectly configured

• Green detector voltage is too low

• Green channel is occupied or compensated incorrectly

• Incorrect emission filter is installed

• The 530 nm signal is outside the acquisition scale

Cell Count Is Higher Than Expected

Possible causes:

• Microsphere volume was under-delivered

• Bead concentration value was entered incorrectly

• Microspheres were excluded from the gate

• Aggregates were excluded as one event

• Sample dilution factor was incorrect

• Cell gate included debris or doublets

Cell Count Is Lower Than Expected

Possible causes:

• Excessive microsphere volume

• Incorrectly high beads/mL value

• Cell loss during washing

• Target cells excluded from the gate

• Cell aggregates counted as single events

• Incorrect original sample volume

• Sample degradation

Results Vary Between Replicates

Possible causes:

• Microsphere settling

• Inconsistent mixing

• Pipetting variation

• Different acquisition delays

• Low event counts

• Gate movement

• Variable flow rate

• Sample heterogeneity

Double-Fluo Beads vs. Other Counting Methods

Double-Fluo Microspheres are particularly useful when users require an internally added counting standard with two fluorescence-identification channels.

Quality Control for Batch Production

Absolute counting microspheres require both material quality control and metrological control.

Recommended release parameters include:

Physical Properties

• Mean particle size

• Particle-size distribution

• Coefficient of variation

• Particle morphology

• Aggregate percentage

• Density, when relevant

• Appearance

• Redispersion performance

Fluorescence Properties

• 488 nm excitation response

• 530 nm emission intensity

• 633 nm excitation response

• 670 nm emission intensity

• Dual-positive percentage

• Green fluorescence CV

• Far-red fluorescence CV

• Green-to-far-red intensity ratio

• Photostability

• Storage stability

Concentration Properties

• Solids content

• Assayed beads/mL

• Particle-count tolerance

• Bottle-to-bottle variation

• Filling-volume accuracy

• Sampling homogeneity

• Stability of assigned concentration

Functional Properties

• Microsphere gate separation

• Recovery during flow cytometry

• Agreement with reference-count samples

• Linearity across cell concentrations

• Within-run precision

• Between-run precision

• Operator reproducibility

• Instrument compatibility

Recommended Incoming Inspection

Reagent manufacturers should consider testing each incoming lot for:

1. Appearance

2. Redispersion

3. Particle size

4. Aggregate level

5. Green fluorescence

6. Far-red fluorescence

7. Dual-positive percentage

8. Beads/mL

9. Flow cytometry recovery

10. Comparison with a qualified reference lot

Reference-Lot Strategy

A qualified reference lot should be retained for comparison with future lots.

New lots can be evaluated using:

• The same cytometer

• The same detector settings

• The same sample

• The same pipette

• The same acquisition template

• The same gating method

• The same calculation software

Batch Documentation

Available or customized documents may include:

• Certificate of Analysis

• Product specification

• Safety Data Sheet

• Particle-size report

• Fluorescence report

• Bead-concentration report

• Stability data

• Lot-comparison data

• Manufacturing change notification

Customization and Bulk Manufacturing

SHBC supports customized fluorescent microsphere development for flow cytometry reagent manufacturers.

Potential customization options include:

• Particle size

• Particle-size distribution

• 488 nm excitation intensity

• 530 nm emission intensity

• 633 nm excitation intensity

• 670 nm emission intensity

• Green-to-far-red intensity ratio

• Solids content

• Beads/mL

• Suspension buffer

• Surfactant system

• Preservative system

• Package volume

• Low-binding formulation

• OEM labeling

• Private-label packaging

Development Supply

Small quantities can be supplied for:

• Instrument compatibility

• Gating development

• Antibody-panel evaluation

• Pipetting studies

• Concentration optimization

• Stability screening

Pilot Production

Pilot lots can support:

• Process transfer

• Filling validation

• Method validation

• Inter-instrument comparison

• Packaging evaluation

• Accelerated stability studies

Bulk Manufacturing

Bulk production may include:

• Lot reservation

• Customer-specific release criteria

• Customized concentration

• Customized packaging

• Long-term supply planning

• OEM and ODM services

• Batch documentation

Storage and Handling

Recommended practices include:

• Store at 2–8°C unless otherwise stated on the product label or COA.

• Protect the product from direct light.

• Do not freeze unless freeze-thaw stability has been validated.

• Mix thoroughly before use.

• Avoid contamination of the stock suspension.

• Use calibrated pipettes.

• Use clean, low-binding tubes and tips.

• Avoid prolonged exposure to elevated temperature.

• Close the bottle immediately after use.

• Follow the lot-specific expiration date.

Commercial counting-bead products are commonly stored at 2–8°C and protected from light.

Before use:

1. Inspect the suspension.

2. Confirm that settled microspheres can be redispersed.

3. Mix according to the validated procedure.

4. Avoid introducing foam.

5. Pipette immediately after mixing.

6. Return the bottle to the recommended storage condition.

Frequently Asked Questions

What are Double-Fluo Absolute Counting Microspheres?

They are fluorescent reference microspheres designed for absolute cell enumeration by flow cytometry.

The microspheres provide fluorescence responses near 530 nm and 670 nm under approximately 488 nm and 633 nm excitation.

What is the solids content?

The product has a solids content of 0.5%.

This corresponds to approximately 5 mg of microsphere material per milliliter when expressed as weight per volume.

Can 0.5% solids be used directly in the counting formula?

No.

The absolute-counting formula requires the lot-specific particle number concentration in beads/mL or beads/μL.

Solids content alone is insufficient.

Why does the product need a lot-specific beads/mL value?

The calculated cell concentration is directly proportional to the assigned microsphere concentration.

Any error in beads/mL produces a corresponding error in the cell-count result.

What lasers are required?

The nominal excitation wavelengths are 488 nm and 633 nm.

Flow cytometers using a 635 nm or 640 nm red laser may also be evaluated.

Which fluorescence channels are used?

The nominal emission channels are approximately 530 nm and 670 nm.

The exact detector filters must be matched to the actual fluorescence spectra.

Can the microspheres be detected using only one channel?

Potentially yes.

However, using both fluorescence channels may improve microsphere identification and gating confidence.

The method should be validated with the intended antibody panel.

Can Double-Fluo Microspheres be used with FITC- or APC-conjugated antibodies?

They may overlap with green- or far-red-emitting antibody conjugates.

Panel compatibility, compensation and gate separation must be evaluated before use.

Can the microspheres be used with whole blood?

They may be evaluated with whole blood and lyse-no-wash workflows.

The lysis reagent, antibody panel, sample volume and cytometer must be validated.

Can they be used with cultured cells?

Yes. They may be evaluated for absolute enumeration of cultured cells, immune cells and other particle suspensions.

How many bead events should be collected?

A minimum of approximately 1,000 bead events can be used as a starting target during method development.

The final requirement should be based on the required precision and expected sample concentration.

Why can I not see the microspheres on FSC and SSC?

The scatter threshold may be too high, or the microspheres may have lower scatter than the target cells.

Reduce the threshold carefully and use the fluorescence channels to identify the microsphere population.

Why do the microspheres settle?

Microspheres have a different density from the suspension medium and may gradually settle during storage.

Mix thoroughly before every aliquot.

Can the particle size be customized?

Particle-size customization may be available according to project quantity and technical requirements.

Can the fluorescence intensity be customized?

Green intensity, far-red intensity and the ratio between the two channels may be discussed for OEM projects.

Can the solids content be customized?

Yes. Different solids contents may be available depending on production quantity and project requirements.

Is bulk supply available?

Yes. SHBC supports evaluation quantities, pilot batches, bulk manufacturing, lot reservation and customized packaging.

Is this a finished diagnostic reagent?

No.

The microspheres are supplied for research use and reagent development. They are not a finished diagnostic product unless separately validated and registered by the customer.

Request a Sample or Bulk Quotation

SHBC supplies Double-Fluo Flow Cytometry Absolute Counting Fluorescent Microspheres for immune-cell enumeration, immunophenotyping research, flow cytometer method development and batch reagent manufacturing.

To request a sample, technical consultation or bulk quotation, please provide:

• Intended application

• Target cell population

• Sample type

• Flow cytometer model

• Available laser wavelengths

• Detector filter configuration

• Antibody panel

• Required particle size

• Required beads/mL

• Required solids content

• Required package size

• Expected annual demand

• Special quality-control requirements

Contact SHBC to evaluate Double-Fluo Microspheres for your absolute cell counting, immune-monitoring, flow cytometry or OEM reagent-development project.