CountBright™ Absolute Counting Beads, for flow cytometry - FAQs

View additional product information for CountBright™ Absolute Counting Beads, for flow cytometry - FAQs (C36950)

13 product FAQs found

使用Live/Dead BacLight细菌活力和计数试剂盒进行流式细胞术实验时，部分细胞好像同时出现红绿两种信号，这些细胞是已经死亡、仍在存活还是即将死亡？

试剂盒中的绿色染料是细胞通透性核酸染色剂，会标记所有细胞。红色染料不能渗透进细胞，只能对膜受损的细胞（死细胞）进行染色。因此，任何具有红色信号的细胞会被认为是死亡的。您的细胞可能会出现有些只有红色信号，有些有红色和绿色信号，有些只有绿色信号的现象。有时红色染料会置换绿色染料。在任何情况下，红色细胞都是死亡的。

另外，绿色染料可能漏进红色通道。进行单色染色，同时在绿色和红色滤光片下检查确定漏光程度。为了避免这种漏光的情况，请使用低浓度的染料；如果可能的话，使用窄带通滤光片。

如何通过流式细胞术对细菌进行计数？

这里有几种办法。我们有两种荧光试剂盒可用于细菌计数：Live/Dead BacLight Bacterial Viability and Counting Kit（货号L34856）；Bacteria Counting kit, for flow cytometry（货号B7277）。另一个选择是Flow Cytometry Sub-micron Particle Size Reference Kit（货号F13839）。

我不太清楚CountBright绝对计数微珠（货号C36950）实验方案中的公式，你们可以帮助我吗？

该公式是让您用计数区域的细胞数除以分析的微珠数。该值随后乘以你们加入的微珠数。该试验方案要求加入50 µL微珠，并且微珠的包装瓶上列出了每50 µL所含的微珠数量 - 你们要乘以该数值（不要除以50）。最后，将得到的结果除以1000即是细胞数/µL。

我用计数微珠进行细胞计数，但在散点图上找不到微珠，该怎么办？

首先检查您的阈值，看它是否设置为前向散射。如果是的话，微珠可能被阈值排除。降低阙值设置后，应该就可以显示出您的微珠了。

我想用流式细胞仪计数细胞，我该怎么做？

可以向样品中添加内部微球计数的标准品来完成流式细胞仪细胞计数。收集到的微球数量已知体积。这使得您可以计算细胞浓度。

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.