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查看更多产品信息 Countess™ II Automated Cell Counter - FAQs (AMQAX1000)
113 个常见问题解答
我们已经证实CellEvent Caspase 3/7 Green Detection Reagent（货号C10423）结合 SYTOX Red Dead Cell Stain （货号S34859）可用于Countess II FL全自动细胞计数仪上从死亡细胞中鉴别凋亡细胞。更多详情，请见应用说明（https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Laboratory%20Instruments/Files/0615/CO210384-CountessIIFL-Apoptosis-AppNote.pdf)。
我们已经确认可在Countess II FL全自动细胞计数仪上使用下列试剂盒：
•LIVE/DEAD Viabilty/Cytoxicity试剂盒（货号L3224）含有钙黄绿素AM和溴乙啡锭二聚体
•ReadyProbes Cell Viability Imaging试剂盒，蓝/绿（货号R37609）
•ReadyProbes Cell Viability Imaging试剂盒，蓝/红（货号R37610）含有NucBlue Live/NucGreen Dead和NucBlue Live/propidium iodide
见此应用说明(https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Laboratory%20Instruments/Files/0415/CO014723-Countess-II-FL-Viability-Appnote_FHR.pdf)中的详情。
可以，Countess II FL仪器与EVOS成像系统使用同样的光立方。Countess II仪器不能使用光立方。
Countess II或Countess II FL仪器可同时通过USB鼠标控制，因此您可以尝试将USB鼠标插入到仪器的任一USB端口，重装软件并进行软件更新。
•U盘必须FAT32格式化；请在进行后续处理前确认这一点。目前，您应该假定所有其他格式都无法工作。
•更新文件必须在U盘根目录下，不能位于文件夹或子文件夹下。
•文件不能以任何方式重命名。
•文件传输过程中不能压缩。在传输过程中必须保证未损坏。
•设备需要几秒时间识别U盘。
•如有必要，通过USB鼠标检查USB端口功能。
把USB格式化为FAT32：
1.将需要格式化的U盘插入到电脑，找到“我的电脑”
2.右键点击U盘，选择格式化
3.选择文件系统FAT32。
4.点击“开始”。
格式化会导致U盘中所有文件丢失，因此在格式化前请确认您已经将重要的文件复制到其他的驱动盘中。格式化后，在更新Countess II或Countess II FL软件之前，需要将软件重新传输到U盘中。
确保细胞被正确聚焦，即确保活细胞中心明亮而死细胞全黑。如果细胞聚焦不当或在屏幕上看上去暗淡，Countess II或Countess II FL仪器就会将它们计为死细胞。如果细胞聚焦良好、有一个明亮的中心，却被计为死细胞，确认它们在适当细胞大小范围内并尝试调节设置。如果细胞在台盼蓝中暴露太久，会影响其活性。因此，为确保获得最好的结果，每次试验应单独准备新的载玻片进行计数。
某些情况下，高度染色的细胞在明场下看起来像是死细胞；这些细胞需要荧光活性染色剂。
有两个主要原因：相应的细胞真的死了；需要调节焦距。某些情况下，高度染色的细胞在明场下看起来像是死细胞；这些细胞需要荧光活性染色剂。
如果您从同一细胞样品中吸取不同的样品量，技术差异可能是吸取和混合操作所致。请使用最近校准过的移液器并确认细胞悬浮液充分混合，在加入10 µL到台盼蓝中之前反复上下吹打。样品与台盼蓝混合后，上下吹打充分混合并立即滴加到Countess载玻片上。
如果您重复计数同一块载玻片，目测检查图像中的细胞都正确计数。确保各次计数之间没有振动和摇动载玻片。可能每一次载玻片插入时观察的视野有轻微的不同，因此根据细胞的浓度和均一性，在同一块载玻片上进行重复计数时将会有一些差异，尽管差异通常低于10%。当计数少量细胞时，很小的视野改变仅仅导致少量的细胞变化却会有很大的影响，所以在高浓度下计数细胞能减少结果波动。
确认仪器上插有已FAT格式化的U盘。
明场下计算细胞浓度时，假定您在台盼蓝中1:1稀释细胞，所以其已经将稀释度计算在内并且将值乘以2，因此显示出来的细胞浓度是在台盼蓝中稀释前的原始细胞浓度。荧光计数显示出的总细胞浓度并没有计算任何稀释度，是载玻片上的细胞真实浓度。
光源调节可控制LED光线强度，相机增益和曝光时间，并可在捕获图像前调节图像亮度和对比度。细胞上样后，右侧将会出现一个调节光源强度的竖条。其往往用于调低亮度，以使背景稍稍黯淡，从而使活细胞/死细胞之间具有较好的对比度。如果仪器上没有光立方，明场的最佳光强度为10-20左右；如果存在光立方，可轻微调高20-30左右，具体因样品而异。
获得图像后，也可以调节细胞用于计数算法的细胞大小范围和细胞亮度。此操作不会改变图片的实际亮度或图片外观，但是可以筛选计数的细胞。按下“捕获”按钮计数细胞后，可以点击“Adjust（调节）”按钮并向一端移动滑动条，确保尺寸、亮度、circularity为最大值，计数所有细胞。您进入到“Adjust（调节）”界面后，可以调节死/活细胞的尺寸、亮度、circularity。确认您在调节界面选择了活/死细胞按钮，从而最大限度放大活/死细胞群的大小和亮度门限。这是细胞出现在图像中却没有被计数的主要原因。最好先为活/死细胞群扩大门限，确保所有细胞都被计数，之后如果您想要排除特定大小和特定亮度的细胞，可减小门限。
屏幕上有一层薄的屏幕保护塑料薄膜，使用前应该去除。
首先按“Capture（捕获）”计数细胞，然后点击“调节”按钮并确保大小、亮度和circularity门限都处于最大值，以便计数所有细胞。进入到“Adjust（调节）”界面后，可以调节死细胞和活细胞的大小、亮度和circularity，来确保您在调节界面同时选择活细胞和死细胞计数按钮，从而将活细胞和死细胞的大小和亮度完全调到了最大值，确保所有的细胞都被计数，之后如果您想要排除特定大小和特定亮度的细胞，可将其减小。
您也可以选择“Default（默认）”用户预置文件来重新将大小、亮度和circularity设定为最大值。
如果孔洞已经完全最大了，CSV应该指出活细胞和死细胞的细胞尺寸最大和最小值为0-60以及亮度的最大和最小值为0–255。
在细胞计数之前降低明场和荧光强度。
首先，确保您的Countess II或Countess II FL仪器上安装了最新版本软件（可点击此处(https://www.thermofisher.com/us/en/home/products-and-services/promotions/countess-software-download.html)下载）。您可能需要手动调节焦距并设定标称焦距，确保自动对焦功能具有一个聚焦到细胞上的设定点。一旦细胞采用0.4%台盼蓝1：1染色并涂在载玻片上，可能需要沉淀细胞约30秒。然后将载玻片放置在仪器上，在让仪器自动对焦之后，您需要通过手动聚焦来刷新明场聚焦。理想情况下，聚焦后活细胞稍微有点亮，而死细胞全部都是黑的，活细胞中心是亮的。您可能想放大来更好的观察细胞，在您拍照前，使用聚焦滑动条手动调节聚焦。一旦聚焦完成之后，您可以点击蓝色“设置”框来设置标称焦距；设置标称焦距会提高自动对焦与以后使用载玻片时的一致性。
确保屏幕上没有会干扰自动对焦并使得样品很难出现在正确的焦平面上的可见气泡或残渣。
启动聚焦的更多指导准则请见此手册(https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/cellanalysis/Files/0914/MAN0010644_Countess%20II.pdf)第20页。
屏幕亮度不均匀可以通过重设光立方盒修复。首先，确保您的仪器上安装有最新版本的Countess II或Countess II FL软件（可点击此处(https://www.thermofisher.com/us/en/home/products-and-services/promotions/countess-software-download.html)下载）。如果软件已经更新，每次仪器重启时，光立方盒应该相应的调整。同时您也可以在设置菜单下，选择“改变光立方”选项，使得光立方重置，之后关闭仪器再打开。如果您没有光立方或者如果您拥有Countess II仪器（没有荧光选项），此过程可能会帮助您解决屏幕亮度不均匀问题。
选择“默认”程序，恢复大小、亮度和circularity门限至最大值。
亮度和大小滑动条确立了成像算法的阙值，使得用户可以同时设置样品亮度和大小的范围。如果样品的参数设定为最大值，所有亮度水平和大小都被计数，因此无法进行后续调节。您可以移动滑动条的末端或者点击滑动条末端的图标，缩小计数的亮度或大小范围。在“调节”屏幕上操作实例如下：在亮度滑块上重复的点击“调光”图标或者手动移动滑块底部，注意绿色亮度杆高度减少。最终压缩的亮度范围可在整个亮度级内滑动调整，从而可仅涵盖暗淡细胞、亮细胞，或根据需要计数的对象调节。对于细胞大小，可采取相同的方式调节。
Countess II或Countess II FL的最新版软件请见此处(https://www.thermofisher.com/us/en/home/products-and-services/promotions/countess-software-download.html)。如果遇到任何问题，请务必浏览网站看是否提供新版软件，因为软件是定期更新的。您也可点击此处(https://www.thermofisher.com/us/en/home/global/forms/countess-fl-automated-instrument-registration.html)注册您的Countess II或Countess II FL仪器，从而在任何软件更新时接到通知。
很遗憾，这个问题没有确切的答案。非细胞透过染料的吸收速率取决于细胞类型、健康状态、营养物、吞食活力等。
台盼蓝染料接触光线后可能会降解，这些污染物可能会促进形成沉淀。经过冷藏或冷冻后，台盼蓝也可能形成橙/红纤维状聚合物。
台盼蓝是活/死细胞鉴别所必需的成分，可区分不同性质的细胞。但如果不需要活力信息，一些细胞自身可能具有足够的对比度，因而可在没有台盼蓝的明场下计数。假定细胞在台盼蓝中1:1稀释，我们在明场下获得就是细胞浓度值；如果细胞未被稀释，您需要将浓度乘以2，才能获得未与台盼蓝混合的细胞的准确浓度。
不能，Countess II和Countess II FL仪器附带一盒盖玻片，不提供台盼蓝染料，因此其需要单独购买（货号T10282）。如果您单独购买一盒盖玻片，会附带台盼蓝染料。
目前为止，我们没发现其他染料可与Countess II和Countess II FL仪器一起用来进行明场活性测定。但只要能够正确安装光立方，即可在Countess II和Countess II FL仪器中观测到多种荧光染色或蛋白。
Countess II和Countess II FL仪器最适用于哺乳动物计数；但是也可以对其他类型细胞进行计数。
我们已经使用默认设置测试了超过20种常用细胞类型，包括原代细胞、PBMCs、昆虫细胞和鱼细胞。某些类型的细胞可能需要调整某些参数，这些参数可由用户自行优化。
如果有两个细胞相互贴在一起，并且每一个都具有充分明确的细胞形态，成像分析固件会将它们区分为两个不同的细胞。
Countess II和Countess II FL不能计数活的精子和快速运动原生动物。
可以，Countess II和Countess II FL仪器可以计数RBC。但我们建议按照1：10,000的比例稀释血液样品。由于它们的染色和台盼蓝染色模式，仪器无法评估RBCs的活力。
可以，Countess II和Countess II FL仪器能够计数PBMCs，但不能区分白细胞类型。
先进的Countess II和Countess II FL仪器的计数运算法能够清楚地分辨成簇细胞中的细胞边界，通常能够精确的计数细胞。
可以，但是您需要采用多个circularity进行试验，直到您发现最适用于自己的细胞类型的那种。
不可以，细菌太小无法从非细胞碎片中鉴别。
可以，在荧光和明明场模式下，仪器都可以计数，允许简单的转染转化率计算并在结果中显示，如‘‘%阳性’’代表目标光立方。
用湿布对仪器表面进行清洁。清洁LCD屏幕时，关闭仪器，切断电源，用一块轻微湿润的软布沾上LCD清洁剂清洁LCD。清洁屏幕时过分用力会损毁LCD屏幕。清洁后立即擦干屏幕。Countess II和Countess II FL仪器没有活动的部件需要维护，没有管道需要清洁，没有缓冲液需要替换。
Countess II和Countess II FL仪器的尺寸非常小（23 cm (w) x 14 cm (d) x 23 cm (h)），重量不超过3.6 kg（不安装光立方）。Countess II FL仪器中不含有光立方，必须单独购买。
USB接口是完全相同的，然而，如果一个USB或外部驱动同时连接到两个端口上，仅仅第一个连接的储存仪器能够被识别。USB鼠标也能使用任一个USB端口。
Countess II和Countess II FL仪器没有内置存储器，因此没有需要消除的文件。文件可以以标准图片格式（JPEG, PNG, TIFF, 或BMP）存储到USB仪器，并可以通过外部任何图片软件打开，或者数据可以以CSV格式存储到USB仪器并通过电子数据表程序打开。
不可以。您可以使用U盘将自己的数据从仪器转移到电脑并用Windows操作系统。
由于数据不需要特殊的软件打开，因此没有合适的电脑软件。文件可以以标准图片格式（JPEG, PNG, TIFF, 或BMP）存储到USB仪器，并可以通过外部任何图片软件打开，或者数据可以以CSV格式存储到USB仪器并通过电子数据表程序打开。仪器的相关软件可以在这里(https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/countess-software.html)下载。
不需要，Countess II和Countess II FL仪器不需要电脑。
可以，Countess II FL仪器不需要光立方，并且如果不需要荧光性，没有光立方也可正常使用。
可以，Countess II FL仪器能与EVOS成像系统使用同样的光立方。Countess II仪器不使用光立方。如果Countess II FL仪器不安装光立方功能就完全如同Countess II仪器，就像一个明视野的细胞计数仪。
Countess II和Countess II FL 全自动细胞计数仪都包括自动聚焦，触摸屏，明明场计数、使用台盼蓝的可视化测试以及使用一次性载玻片。Countess II FL全自动细胞计数仪同时能适用于重复使用的玻璃载玻片和基于EVOS LED光立方的荧光检测。
这取决于细胞使用的荧光染色的类型。台盼蓝是一个细胞膜非通透性的染料且可以淬灭荧光。其可以淬灭活细胞表面的荧光染色或死细胞内部荧光染色。
Countess II和Countess II FL仪器预先校准过的。您不需要校准仪器。
在使用0.2%台盼蓝的条件下，基于染料的排斥性。台盼蓝被活细胞排斥，能进入死细胞，从而使死细胞胞内的蛋白质被强烈的染色。
下列的细胞系和原代细胞已经通过测试：
细胞系：HEK 293, A431, CHO-M1, CHSE, COLO-205, COS-7, HeLa, HepG2, HL-60, J774(MMM), Jurkat, K-562, MCF-7, MRC-5, NIH/3T3, PC-12, SF-21, U266, 和U2OS
原代细胞：人类脂肪组织来源干细胞, HASMCs, HPAECs, HPASMCs, HUVECs, C2C12, RBCs,和PMBCs
我们在持续测试更多细胞。
当一个实验工作者使用玻璃血球计数板和显微镜手动计数细胞时，单个实验的重复计数的变化率一般为10%或更多。
Countess II和Countess II FL仪器的用户观测到的重复计数的变化率通常应该<5%。
在明场模式下计数，Countess II和Countess II FL仪器自动默认设定1：1与台盼蓝稀释。在荧光模式下，没有这样默认设定。
Countess II仪器被设计用于读取浓度范围从1 x 10E4到1 x 10E7细胞/mL的细胞样本。
每个样品花费的时间为10秒。
Countess II和Countess II FL仪器能够检测到微粒/细胞的范围为4–60 μm。大小在7–60 μm范围的细胞可以在明场下计数和评估细胞活力。
Two things may be happening. Manual hemocytometer counting can be fraught with error, due to user bias and calculation errors. If the cells are clumping, however, the Countess counter may count the clump as one cell (although usually it can discriminate). Look at the images from the Countess counter and the circles it draws over counted cells to see if this is the issue. If so, you can increase the sensitivity of the instrument by adjusting the parameters.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
There are a couple of possibilities:
First, check to see if the settings have been changed (size, circularity and brightness). Another user might have changed the parameters.
Second, make sure your cells are within the size range for the assay (4–60 µm).
Finally, carefully focus the samples.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
What you see on the screen is what is counted. Regions that are not visible are not counted. So if the cells are circled correctly, the cell count should be correct.
Open up the histogram window when setting gates, so that you can see what is included and excluded from the count. It is also a good idea to check the histogram just to make sure any gates have not been set incorrectly even if you are not adjusting them. Be sure to set the same gates for both live and dead cells. You can save gates as a profile so that they are applied the same way to all the samples. Brightfield viability is only going to be accurate for cells with diameters >7 µm, but the instrument will see cells as small as 1 µm. So, if you have a lot of cells with diameters lesser than ~5 µm, we recommend setting a gate on both live and dead cells to remove these small cells from the count and improve the live percentage. Otherwise, these small cells will be counted as dead regardless of whether they are dead or live.
Note: If you do see a lot of 1-2 µm counts (seen in the histogram), it could be due to precipitates in the trypan blue stain, which is common. The three approaches below may help minimize trypan blue precipitate in your cell counting samples:
- Sterile filtration of trypan blue using 0.3 micron filter
- Centrifugation or passive settling of trypan blue solution (avoid mixing or vortexing trypan stock solutions)
- Gentle warming of the 1 mL trypan blue solution to 37 degrees C for 10 mins
The following are parameters to keep in mind to ensure accurate viability determination:
- The Countess II must be able to find one pixel in the center of the cell that is brighter than the edge pixels to be counted as a live cell. So focusing through the widest diameter of the cell for small cells is important.
- Contrast just needs to be a shade of grey, not super black or white for the Countess II to see the cells well.
- Let the cells settle for about 20 seconds before counting (especially for small cells), so that they are all in the same focal plane on the bottom of the slide.
- Be sure that your total cells are less than 1 X 10E7 cells/mL. A good concentration range is about 100-300 cells visible on the screen. If you want, you can find actual numbers of cells counted in the .csv file. If cells are too confluent or if a lot of small debris is present, the Countess II might have a hard time figuring out what is background and be unable find the cells correctly. Clumping is normally not a problem as long as all the cells are in the same focal plane. If no gates are applied and cells are still not circled correctly, we recommend diluting the sample.
To set the nominal focus, please complete the following steps:
- Put in a slide with cells stained with Trypan Blue stain.
- Click the focus icon at the bottom of the count screen; you may also zoom in if desired.
- Use the focus slider to find a good focus. A good focus would be one where your live cells are showing bright centers with defined edges and your dead cells are showing totally dark.
- Once you have a good focus, tap the "set" box on the top of the focus slider.
- Reinsert the slide and allow the autofocus to work from the new nominal focus point. If the nominal focus is not set near a good focus for your cells, the autofocus may be looking in the wrong range and will not work correctly.
- Press "count".
Yes, the instrument multiplies the count by 2 to correct for the 1:1 trypan blue dilution.
Unfortunately, the Countess II Automated Cell Counter does not work well with yeast cells. This instrument can detect cells as small as 4 µm but can only accurately determine viability in brightfield for cells 7 µm and larger. Yeast cells are just below the cut-off for determining viability.
We have validated CellEvent Caspase 3/7 Green Detection Reagent (Cat. No. C10423) in combination with SYTOX Red Dead Cell Stain (Cat. No. S34859) for discriminating apoptotic cells from dead cells using the Countess II FL Automated Cell Counter. For further details, please see this Application Note (http://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Laboratory%20Instruments/Files/0615/CO210384-CountessIIFL-Apoptosis-AppNote.pdf).
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We have validated the following kits for use on the Countess II FL Automated Cell Counter:
LIVE/DEAD Viabilty/Cytoxicity Kit (Cat. No. L3224) containing calcein AM and ethidium homodimer-1
ReadyProbes Cell Viability Imaging Kit, Blue/Green (Cat. No. R37609)
ReadyProbes Cell Viability Imaging Kit, Blue/Red (Cat. No. R37610) containing NucBlue Live/NucGreen Dead and NucBlue Live/propidium iodide
See this Application Note for details - https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Laboratory%20Instruments/Files/0415/CO014723-Countess-II-FL-Viability-Appnote_FHR.pdf.
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Yes, the Countess II FL instrument uses the same light cubes as the EVOS imaging systems. The Countess II instrument does not use light cubes.
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The Countess II or Countess II FL instrument can also be controlled by a USB mouse, so you can try reinstalling the software by plugging a USB mouse into either USB port on the instrument and use the mouse to perform the software update.
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The drive must be FAT32 formatted; verify this before proceeding. At present, you should assume that all other formats will not work.
The update file must sit on the top level of the USB drive, not within a folder or subfolder.
File cannot be renamed in any way.
File cannot be zipped or compressed during distribution. It must be uncorrupted during transfer.
The device takes a few seconds to recognize the USB drive.
If needed, check that the USB port is functional by testing a USB mouse.
To format a USB to FAT32:
Insert drive requiring reformatting into a PC, find dive in “My computer”
Right click on USB drive, select Format.
Select file system, FAT32.
Click Start.
Reformatting will result in the loss of all files from the USB drive, so ensure that you have copies of any important files on another drive prior to reformatting. After reformatting, the software should be retransferred onto the USB drive prior to updating the Countess II or Countess II FL software.
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Ensure that the cells are focused correctly so that live cells have bright centers and dead cells are dark throughout. If the cells are not well focused and look dark on the screen, the Countess II or Countess II FL instrument will count them as dead cells. If cells are well focused, have bright centers, and are being counted as dead, confirm that they are within the appropriate cell size range and try adjusting the settings. If cells are exposed to trypan blue for a long period of time, viability could be affected so the slide should be prepared and counted fresh each time for best results.
In some cases, heavily pigmented cells will also look dead in brightfield; such cells may require fluorescent viability stains.
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There are two predominant reasons for this: either the cells in question are truly dead or the focus should be adjusted. In some cases, heavily pigmented cells will also look dead in brightfield; such cells may require fluorescent viability stains.
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If you are pipetting different samples from the same cell sample, the variability could be due to pipetting or mixing. Use recently calibrated pipettors and make sure that the cell suspension is well mixed by pipetting up and down several times before you add 10 µL to trypan blue. Once the cell sample is mixed with trypan blue, it should also be well mixed by pipetting up and down and loaded into the Countess slide right away.
If you are counting replicates of the exact same slide, visually inspect that all cells are counted correctly in the image. Ensure that you do not shake or agitate the slide between counts. There may be a slightly different field of view each time a slide is inserted, so depending on the concentration and uniformity of the cells, there will be some variability when performing replicate counts of the same slide, although it should be less than 10%. When counting fewer cells, a small field of view change for only a small number of cells can have a larger affect, so variability can be reduced by counting cells at a higher concentration.
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Ensure that a FAT-formatted USB drive is inserted in the instrument.
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The brightfield cell concentration assumes that you have diluted the cells 1:1 in trypan blue, so it already takes this dilution into account and multiples the values by 2 so that the cell concentration displayed is the original cell concentration before dilution in trypan blue. The total cell concentration displayed after a fluorescent count does not take any dilution into account, so it is the actual cell concentration in the slide.
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The light source adjustment controls the LED intensity, camera gain, and exposure time, and it is used for adjusting the image brightness and contrast before you capture the image. Once the cells are inserted, there will be a bar on the right hand side to adjust the light source intensity. Often it helps to turn it down so that the background is slightly darker, which allows better contrast between live and dead cells. Optimal light intensity in brightfield is usually around 10-20 if there are no light cubes in the instrument, and slightly higher around 20-30 if there are light cubes present, but this could vary depending on the sample.
Once the image is captured, you can also adjust the range of cell size and cell brightness that the counting algorithm will count. This doesn't change the actual brightness of the image or how it looks, but determines which cells will be counted. Once you press “Capture” to count the cells, you can hit the “Adjust” button and make sure that the size, brightness, and circularity gates are all maximized by maximizing the slider bars to include all of the cells in the count. When you go into the “Adjust” screen, you can adjust the size, brightness, and circularity for both live and dead cells, so make sure that you select both the live and dead buttons in the adjust screen so that you are completely maximizing the size and brightness gates for both the live and dead cell population. This is mainly an issue if there are cells that appear in the image, but aren't being counted. It's best to expand the gates first, make sure all of the cells are counted, and then narrow them if you want to exclude cells of a certain size or certain brightness.
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The screen has a thin plastic film screen protector that should be removed before use.
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Once you press “Capture” to count the cells, hit the “Adjust” button and make sure that the size, brightness, and circularity gates are all maximized to include all of the cells in the count. When you go into the “Adjust” screen, you can adjust the size, brightness, and circularity for both live and dead cells, so make sure that you select both the live and dead buttons in the adjust screen so that you are completely maximizing the size and brightness gates for both the live and dead cell population. It's best to expand the gates first, make sure all of the cells are counted, and then narrow them if you want to exclude cells of a certain size or certain brightness.
You can also select the “Default” user profile to restore the size, brightness, and circularity gates to their maximum. If the gates are fully maximized, the CSV should indicate 0-60 for cell size min and max and 0-255 for brightness min and max for both live and dead cells.
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Decrease the brightfield and fluorescence light intensity before counting the cells.
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First, make sure you have the most recent version of the Countess II or Countess II FL software installed on the instrument (it is available here). You may need to manually adjust the focus and set the nominal focus so that the autofocus will have a set point to focus on the cells. Once cells are stained 1:1 in 0.4% trypan blue and placed in the slide, you may want to let the cells settle for around 30 seconds. Then place the slide in the instrument and after you let the machine autofocus, you may need to refine the brightfield focus with the manual focus. Ideally the live cells should be slightly under focused, where the centers are bright compared to the dead cells that are dark throughout. You may want to zoom in so that you can better see the cells, then manually adjust the focus using the focus slider bar before you hit capture. Once the cells are focused, you can tap the blue “set” box to set the nominal focus; setting the nominal focus will improve autofocus consistency with future slides.
Make sure there are no bubbles or debris visible on the screen that could interfere with the autofocus and make it more difficult to get the sample in the correct focal plane. You can find some more guidelines for focusing starting on page 20 of the manual.
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Uneven illumination on the screen may be fixed by resetting the light cube tray. First, make sure you have the most recent version of the Countess II or Countess II FL software installed on the instrument (it is available here). If the software is updated, the light cube tray should readjust each time the instrument is power cycled. You can also select the “change light cubes” option under the settings menu, allow the light cubes to reset, and then turn the instrument off and back on. This process may help with uneven screen illumination even if you do not have light cubes present or if you have the Countess II instrument (without the fluorescence option).
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Select the “Default” profile to restore the size, brightness, and circularity gates to their maximum.
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The brightness and size sliders establish the thresholds for the imaging algorithm, allowing users to set both the brightness and size ranges for the sample. If the parameters are at their maximum settings, all brightness levels and sizes are counted, so they cannot be further adjusted. To narrow the brightness or size range for the count, you can move the ends of the slider bar or you can hit the icons at the end of the slider bar to adjust them. To demonstrate this while on the ‘Adjust' screen, repeatedly tap the ‘dim' icon on the brightness slider or manually move the bottom end of the slider and watch the green brightness bar decrease in height. The resulting narrowed brightness range can be slid across the brightness scale to include only dim cells, only bright cells, or something in between depending on what is to be included in the count. The same adjustments can be performed for cell size.
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The most recent version of the Countess II or Countess II FL software is available here (https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/countess-software.html). There will be no more software updates to Countess II/IIFL.
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Unfortunately, there is no definitive answer to this question. The rate at which the cell-impermeant dye is absorbed depends on the cell type, their state of health, nourishment, engulfment activity, etc.
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Exposure to light may degrade the dye and these contaminants may promote precipitation. Trypan blue can also form orange/red fibrous aggregates if exposed to refrigeration or freezing temperatures.
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Trypan blue is necessary for live/dead discrimination and it provides contrast for the cells, however if viability information is not required, some cells may have enough contrast on their own to be counted in brightfield without trypan blue. The cell concentration value obtained in brightfield assumes the cells are diluted 1:1 in trypan blue, so if the cells are not diluted, you will need to divide the concentration by 2 to get an accurate concentration for cells that are not mixed with trypan blue.
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No, the Countess II and Countess II FL instruments are provided with a box of slides, but are not provided with trypan clue stain so it will need to be purchased separately (Cat. No. T10282). If you purchase a box of slides separately, the slides will come with trypan blue stain.
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Erythrosine B (0.04%) can be used a a substitute for Trypan Blue in the Countess II and Countess II FL instruments.
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The Countess II and Countess II FL instruments were optimized for mammalian cell counting; however, counting other cell types may be possible.
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We have tested over 20 commonly used cell types, including primary cells, PBMCs, insect cells, and fish cells, using the default settings. Specific cell types could require some parameter setting adjustments, and those may be optimized by the user.
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If there are two cells attached to each other with enough circular definition for each, the image analysis firmware will distinguish them as two different objects.
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The Countess II and Countess II FL instruments do not count live sperm cells or other fast-moving cells such as protozoa.
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Yes, the Countess II and Countess II FL instruments can count RBCs. However, we recommend diluting the blood sample approximately 1:10,000. The instrument cannot assess the viability of RBCs due to their pigmentation and trypan blue staining pattern.
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Yes, the Countess II and Countess II FL instruments can count PBMCs. However, they cannot differentiate between white blood cell types.
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The advanced counting algorithms of the Countess II and Countess II FL instruments can clearly identify cell boundaries within clumps of cells, typically resulting in accurate cell counts.
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Yes, but you may need to experiment with several circularity settings until you find the one that is best for your cell type.
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No, bacteria are too small to be distinguished from non-cell debris.
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Yes, the instrument can count in both fluorescence and brightfield modes, allowing simple calculation of transfection efficiency that is shown on the results screen as “% positive” for the light cube of interest.
Clean the surface of the instrument with a damp cloth. To clean the LCD screen, turn off the instrument, disconnect the power cable, and clean the LCD screen with a soft cloth lightly moistened with LCD cleansing detergent. Cleaning the screen with excessive force can damage the LCD screen. Wipe the screen dry immediately. The Countess II and Countess II FL instruments have no moving parts to maintain, no tubes to clean, and no buffers to replace.
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The Countess II and Countess II FL instruments have a very small footprint (23 cm (w) x 14 cm (d) x 23 cm (h)) and weigh about 3.6 kg (without light cubes installed). Light cubes are not included with the Countess II FL instrument; they must be purchased separately.
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The USB ports are identical, however if a USB or external drive is connected to both ports at the same time, only the first storage device connected will be recognized. A USB mouse can also be used in either of the USB ports.
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Countess II and Countess II FL instruments have no internal memory, so there are no files that need to be erased. Files can be saved to the USB as standard image files (JPEG, PNG, TIFF, or BMP) to be opened by any external imaging program, or data can be saved to the USB in a CSV file to be opened by a spreadsheet program.
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No. You can use a USB drive to transfer your data from the instrument to a computer that uses a Windows operating system.
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There is no computer software available since the data does not require any special software to open. Files can be saved to the USB as standard image files (JPEG, PNG, TIFF, or BMP) to be opened by any external imaging program, or data can be saved to the USB in a CSV file to be opened by a spreadsheet program. The software for the instruments may be downloaded here (https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/countess-software.html).
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No, the Countess II and Countess II FL instruments do not require a computer.
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Yes, the Countess II FL instrument does not require light cubes and can be used without them if fluorescence is not required.
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Yes, the Countess II FL instrument uses the same light cubes as the EVOS imaging systems. The Countess II instrument does not use light cubes. The Countess II FL instrument without light cubes installed will function exactly like the Countess II instrument-as a brightfield cell counter.
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Both the Countess II and Countess II FL Automated Cell Counters include autofocus, a touch screen, brightfield counting, and viability testing using trypan blue, and use disposable slides. The Countess II FL Automated Cell Counter can also accommodate a reusable glass slide and fluorescence detection using EVOS LED-based light cubes.
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It depends on the type of fluorescent stain used on the cells. Trypan blue is a cell-impermeant chromophore that can quench fluorescence. It may quench fluorescent staining on the surface of live cells or internal fluorescent staining in dead cells.
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The Countess II and Countess II FL instruments come precalibrated. You do not have to calibrate the instruments.
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It is based on dye exclusion using 0.2% Trypan Blue dye. Trypan Blue is excluded by the live cells and enters the dead cells; this results in intense staining of the intracellular proteins.
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The following cell lines and primary cells have been tested:
- Cell lines: HEK 293, A431, CHO-M1, CHSE, COLO-205, COS-7, HeLa, HepG2, HL-60, J774(MMM), Jurkat, K-562, MCF-7, MRC-5, NIH/3T3, PC-12, SF-21, U266, and U2OS
- Primary cells: Human adipose tissue-derived stem cells, HASMCs, HPAECs, HPASMCs, HUVECs, C2C12, RBCs, and PMBCs
We are continually testing additional cell types.
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When an experienced worker manually counts cells using a glass hemocytometer together with a microscope, count-to-count variability of a single sample is commonly 10% or more. Users of Countess II and Countess II FL instruments should typically see <5% count-to-count variability.
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When counting in brightfield mode, the Countess II and Countess II FL instruments automatically assume a 1:1 dilution has been made with trypan blue. The same assumption is not made when in fluorescence mode.
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The Countess II instruments are designed to read samples with concentrations in the range of 1 x 10E4 cells/mL to 1 x 10E7 cells/mL.
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The time taken is ˜10 seconds per sample.
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The Countess II and Countess II FL instruments can detect particles/cells ranging from 4 - 60 µm. Cells ranging from 7 - 60 µm can be counted and assessed for viability with brightfield viewing.
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