Molecular Probes™ CountBright™ Absolute Counting Beads, for flow cytometry

Product Code.	Quantity	unitSize
11570066	5 mL	5mL

Product Code. 11570066 Supplier Molecular Probes™ Supplier No. C36950

Description

CountBright™ Absolute Counting Beads, for flow cytometry

CountBright™ absolute counting beads are a calibrated suspension of microspheres that are brightly fluorescent across a wide range of excitation and emission wavelengths (UV to 635 nm excitation and 385-800 nm emission). CountBright™ absolute counting beads are mixed with the cell sample and assayed via flow cytometry. By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample can be calculated. Because CountBright™ beads are mixed in the test sample, absolute cell counts using this single-platform method are more accurate and less complicated than cell concentration determined using multiple-platform testing. CountBright™ absolute counting beads can be used with any sample type, including no-wash/lysed whole blood.

View information about all flow cytometry cell counting beads.

Specifications

• Diameter (Metric): 7 μm
• Content And Storage: Contains 1 bottle of CountBright™ absolute counting beads (5 mL). Store in refrigerator (2°C to 8°C) and protect from light.
• Emission: any lasers (UV to 633/635)
• Format: Solution
• For Use With (Equipment): Flow Cytometer
• No. of Tests: 100
• Quantity: 5 mL
• Detection Method: Fluorescence
• Excitation Wavelength Range: UV to 635/385 to 800 nm
• Shipping Condition: Room Temperature
• Product Line: CountBright
• Product Type: Cell Counting Bead

Frequently Asked Questions (FAQs)

With the CountBright Plus Absolute Counting Beads/CountBright Absolute Counting Beads, instead of a tight cluster of beads on the scatter plot, I am seeing the beads cover a broad area. What is causing this?

Prior to taking an aliquot, make certain to thoroughly mix the beads. This may also be caused if the beads were ever frozen or the stock solution has any microbial contamination.

Which buffers can I use with the CountBright Plus Absolute Counting Beads and CountBright Absolute Counting Beads?

You may use any physiological buffer or media in a pH range from pH 4 to 10. Avoid extremes of pH and high salt concentrations.

How are CountBright Plus Absolute Counting Beads different from CountBright Absolute Counting Beads?

Here are the differences:

CountBright Plus Absolute Counting Beads:
Bead Size: 4 µm
Excitation Range: 385 - 810 nm
Emission Range: 385 - 860 nm

CountBright Absolute Counting Beads:
Bead Size: 7 µm
Excitation Range: 350 - 635 nm
Emission Range: 350 - 810 nm

When using the Live/Dead BacLight Bacterial Viability and Counting Kit, for flow cytometry, some cells seem to have both red and green signal. Are these cells dead or alive or dying?

The green dye in the kit will label all the cells as it is a cell-permeant nucleic acid stain. The red dye is not cell permeant, and will only stain the cells with compromised membranes (dead cells). Therefore, any cells with red signal will be considered dead. It is possible that you will have some cells that are only red, some that are red and green, and some that are only green. Sometimes the red dye will displace the green dye. In any case, any red cells are dead.

Also, the green dye emission may bleed through into the red channel. Do a single-color staining and examine under both green and red filter sets to determine the level of bleedthrough. To avoid this bleedthrough, use a lower concentration of dye, and, if possible, use narrow bandpass filters.

How can I count my bacteria by flow cytometry?

There are several options. We have two fluorescence based kits that are useful for bacterial counting: Live/Dead BacLight Bacterial Viability and Counting Kit, for flow cytometry (Cat. No. L34856) and Bacteria Counting kit, for flow cytometry (Cat. No. B7277). Another option is the Flow Cytometry Sub-micron Particle Size Reference Kit (Cat. No. F13839).

I am confused by the formula in the protocol for CountBright Absolute Counting Beads (Cat. No. C36950). Can you please help?

The formula has you divide the number of cells in the region you want to count by the number of beads analyzed. This value is then multiplied by the number of beads you added. The protocol calls for adding 50 µL of beads, and the vial of beads lists the number of beads per 50 µL- this is the number that you multiply by (do not divide by 50.) Finally, you divide by 1,000 to get your result as the number of cells/µL.

I am using counting beads to count cells, but I cannot find the beads on my scatter plots. What do I do?

The first thing to do is check your threshold and see if it is set on forward scatter. If so, the beads are probably being excluded by the threshold. Reducing the threshold setting should reveal your beads.

I want to count my cells using flow cytometry. How do I do this?

Cell counting using flow cytometry can be accomplished by adding an internal microsphere counting standard to the flow cytometric sample. The number of reference beads that are collected reflects a known volume. This allows you to calculate cell concentration.

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