LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit, for flow cytometry - FAQs

View additional product information for LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit, for flow cytometry - FAQs (L34856)

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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）。

我很难从仪器背景信号中区分我的细菌，该如何优化信号？

电子背景信号是由系统光学元件收集的杂散光和/或低水平电子信号造成的。背景信号可以通过正确设置仪器阈值和光电倍增管（PMT）电压而消除或降低。从其它微小颗粒和电子背景信号中区分细菌群落的最简单方法是用荧光染料标记细菌，然后使用该荧光来识别目标群落。SYTO 9是能够标记所有细胞的出色染料，可通过FITC通道检测。

我可以在流式细胞仪上看到哪些细菌参数?

您可以用BacLight Green细菌染色剂（货号B35000）或BacLight Red细菌染色剂（货号B35001）等常规染色剂进行细菌染色。你们可以看到革兰氏特性（货号L7005），细胞存活率（货号L7007、L7012和L13152），细胞计数（货号L34856和B7277）和细胞活力。细胞活力可以通过膜电位法（货号B34950）或代谢法（货号B34954和B34956）测定。

我可以使用LIVE/DEAD BacLight 细菌活性和计数试剂盒（货号L34856）对细菌染色，从而检测人细胞样品中的细菌吗？

效果不好，因为该试剂盒中的染料也会染色哺乳动物细胞。您可以根据散射特性区分细胞，但是染色剂无法验证您的选择。

我可通过流式细胞仪观察细菌和酵母么？

可以。流式细胞仪可用于：

•计数检测：用于流式细胞仪的细菌计数试剂盒（货号B7277）或LIVE/DEAD BacLight细菌活性和计数试剂盒（货号L34856）
•活性/活力检测：LIVE/DEAD BacLight 细菌活性试剂盒（货号 L13152）
•膜电位：BacLight细菌膜电位试剂盒（货号B34950）
•酵母活性/活力实验：用于流式细胞仪（货号L34952）的LIVE/DEAD FungaLight酵母活性试剂盒，FungaLight酵母CFDA, AM/碘化丙啶活力试剂盒（货号F34953）。

When using the LIVE/DEAD BacLight kit for bacterial viability, with SYTO 9 and propidium iodide (PI), why are some cells detected with both dyes? Shouldn't live cells be green and dead cells be red?

SYTO 9 dye will enter all cells, live or dead. PI only enters cells with compromised membranes (dead cells or damaged cells). First, perform single color staining and examine under both filter sets. Longpass filters or the use of too much dye may result in excessive bleedthrough of the green dye emission into the red channel and the emission of PI in the green channel. Use narrower bandpass filters if possible, and use lower concentrations of the dye. Some live cells may take up PI by engulfment; avoid extended incubation times with the dye. Apply PI to a live cell culture and optimize incubation times to limit engulfment.

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

I want to use propidium iodide and SYTO 9 to do LIVE/DEAD testing of a bacterial sample. Will anaerobic conditions adversely affect the assay?

Oxygen content should not affect the binding of propidium iodide and SYTO 9 to nucleic acids. SYTO 9 will label all cells, and propidium iodide will label only dead cells or cells with a compromised membrane.

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

I have a LIVE/DEAD BacLight Bacterial Viability kit that has SYTO 9 and propidium iodide in it. Will I be able to stain eukaryotic cells that have engulfed bacteria and determine if the bacteria are alive or dead using this kit?

Unfortunately, no. SYTO 9 will label the nuclei of live or dead cells, including the eukaryotic cells. Propidium iodide is cell impermeant, and will only enter dead cells. If the eukaryotic cells are dead, they will label with propidium iodide as well. If the eukaryotic cells are alive, propidium iodide will not be able to enter and thus will not label the bacteria inside, whether the bacteria are alive or dead. We are not aware of any way to do a viability assay of bacteria once they have been engulfed by cells.

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 having a hard time separating my bacteria from instrument noise. How can I optimize my signal?

Electronic noise is due to stray light collected by the system optics and/or low-level electronic signals. Noise may be eliminated or reduced through correct setup of instrument threshold and photomultiplier tube (PMT) voltage. The easiest way to differentiate a bacterial population from other small particles and electronic noise is to label the bacteria with a fluorescent stain and use the fluorescence emission to identify the population of interest. SYTO 9 is a great stain to label all cells and is detected in the FITC channel.

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

What bacterial parameters can I look at by flow cytometry?

You can stain bacteria with a general stain such as BacLight Green Bacterial Stain (Cat. No. B35000) or BacLight Red Bacterial Stain (Cat. No. B35001). You can look at gram character (Cat. No. L7005), cell viability (Cat. Nos. L7007, L7012, and L13152), cell count (Cat. Nos. L34856 and B7277), and cell vitality. Cell vitality can be measured by membrane potential (Cat. No. B34950) or by metabolism (Cat. Nos. B34954 and B34956).

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

Can I use a bacterial stain such as the LIVE/DEAD BacLight Bacterial Viability and Counting Kit (Cat. No. L34856) to detect bacteria in my human cell sample?

This will not work well because the dyes in this kit will also stain mammalian cells. You can probably distinguish cells based on their scatter properties, but the stain will not confirm your selection.

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

Can I look at bacteria and yeast on a flow cytometer?

Yes, you can. We offer:

-Counting assays: Bacteria Counting Kit, for flow cytometry (Cat. No. B7277) or LIVE/DEAD BacLight Bacterial Viability and Counting Kit, for flow cytometry (Cat. No. L34856).
-Viability/vitality assays: LIVE/DEAD BacLight Bacterial Viability Kit (Cat. No. L13152).
-Membrane potential: BacLight Bacterial Membrane Potential Kit (Cat. No. B34950).
-Yeast viability/vitality assays: LIVE/DEAD FungaLight Yeast Viability Kit, for flow cytometry (Cat. No. L34952), FungaLight Yeast CFDA, AM/Propidium Iodide Vitality Kit (Cat. No. F34953)

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