Celigo image cytometer
| Question | Answer |
|---|---|
| Can the Celigo be used as a plate reader? | The Celigo cannot be used to measure fluorescence, absorbance, or luminescence levels in the supernatant of a well, so it cannot be used as a traditional plate reader. However, it does have the analysis capability to measure the total fluorescence intensity imaged within the cell layer. |
| Can the Celigo be used for Luminescence detection? | No, the current Celigo configuration does not allow the detection of luminescence assays. |
| Can the Celigo do neuron outgrowth or other branching assays, as well as tube formation? | No, the current algorithms are not designed for neuronal analysis, but some neurobiologists use it to capture the whole area to get a birds-eye view of the slide. It’s much faster than a traditional fluorescent microscope that is used by neurobiologists. |
| How do I get started using the Celigo? | We recommend reading through the Quick Start Guide that is shipped with your instrument. If you are looking for more assistance, please contact our Support Team via cellc-support@revvity.com |
| How do we adjust the Celigo software to default to selected analysis settings for a particular project? | The Projects are based on saved experiment settings. Once a project is created you cannot change which saved experiment it refers to. However, if you change the save “Experiment” settings (by overwriting them) the behavior of the project will change accordingly. If you would like to change what the project does, and not change the “Experiment” settings it is based on (because they are used elsewhere for example) the best course of action is to save a new “Experiment” followed by generating a new “Project”. You can then delete the old “Project” and rename the new “Project” to the old “Projects” name. |
| I would like to connect a Celigo PC to my organization’s network. How can I do this? | You will need to contact our Support Team via cellc-support@revvity.com. Each time you change a domain or a computer name, login permissions are broken between the Celigo/Cellaca and the SQL database. Support can bypass this to ensure login permissions are maintained for the new user or user group on the domain. |
| Is the Celigo temperature controlled? | No, but it is around 10 degrees warmer than the room temp it is running in, so if RT is 25°C, then its ~ 35°C inside instrument. |
| What is the cost per sample for a Celigo run? | This depends on several factors, e.g. which assay you are running, what kind of plates are you using? Contact our Support Team for assistance via cellc-support@revvity.com |
| What types of 3D spheroid assays can be performed on the Celigo? | Growth and inhibition studies (endpoint and kinetic studies), migration on ECM and HUVEC cells and inhibition studies (endpoint and kinetic studies), invasion and inhibition studies (endpoint and kinetic studies), tissue invasion and inhibition studies (endpoint and kinetic studies), cell-mediated cytotoxicity, viability staining using CAM/PI, apoptosis, hypoxia |
Cellaca MX and PLX instruments
| Question | Answer |
|---|---|
| Can we use a 96-well plate with the Cellaca? | The Cellaca systems are only compatible with Cellaca plates, which are available in two formats (3×8) and (2×12). |
| Can we use a partial plate on the Cellaca? | Yes. Be sure to mark the wells that are used for future reference. |
| How do I get started using the Cellaca MX? | We recommend reading through the Quick Start Guide that is shipped with your instrument. If you are looking for more assistance, please contact our Support Team via cellc-support@revvity.com |
| How many images does the Cellaca capture for its counting? | The Cellaca MX system takes 1 image for each channel per well, so if you are loading a full 3×8 Cellaca plate, and imagine in the brightfield, and two fluorescent channels, the Cellaca will take (1 image) * (3 channels) * (24 wells) = 72 images. |
| How often should calibration beads be run on the Cellaca? | The beads are considered verification beads rather than calibration beads. The calibration of the instrument is something that must be done by one of our field service engineers. The verification beads are to be used at your discretion. They are manufactured at a known concentration range, so you know that if your results fall in that range, your instrument is working. |
| What are the dimensions of the Cellaca? | The height, width, and depth of the Cellaca system are 13 in (33cm), 13 (33cm), and 16 (41cm), respectively. |
| Why does my Cellaca preview not work in V 2.0.1? | You need to make sure the Windows resolution of your computer is set to 100% and 1920×1080. From the Start panel, go to Settings -> Display -> Scale and Layout, and set these values. If the issue persists, please contact cellc-support@revvity.com |
Cellometer cell counters
| Question | Answer |
|---|---|
| Can I open up my Cellometer instrument and clean its components? | No, we ask that customers do not take the cover off their instruments. The instruments do not require cleaning, calibration, or any standard, routine maintenance. If the instrument needs to be fixed internally, please contact cellc-support@revvity.com |
| Can I use the Cellometer to count adipocytes? Can I measure viability? | Yes. The first thing to note is that when working with adipocytes, you will need to use our CHT4-PD300 slides. These slides are specifically designed for larger cells. Secondly, you will need to use a combination of Calcein-AM and propidium iodide (PI) in order to measure viability. Calcein-AM is a stain that will fluoresce green in metabolically active cells. The PI, however, can only enter cells with compromised cell membranes (dead/dying cells) and will fluoresce red. The cells that fluoresce green indicate that they are metabolically active and alive, and the cells that fluoresce red indicate that they are dead cells. The catalog number for this kit is CSK-0118. |
| Can slides with the grid on them (CP2 slides) be used in the Cellometer? | No, the CP2 slides are made to be used as a disposable hemocytometer and used with a standard microscope. We have multiple slides at various configurations specifically designed to be compatible with the Cellometer instruments. |
| Can the Cellometer instruments measure irregularly shaped cells? | Yes. The Cellometer software can be set to count cells of irregular shapes within the parameter settings of each assay. |
| Can the Cellometer software count PBMCs? | Yes, the Cellometer Ascend, Cellometer Auto 2000, Cellometer K2, and Cellometer Spectrum are recommended for analysis of PBMCs from heterogeneous samples. Cultured PBMCs can be analyzed with the Cellometer Auto T4. |
| Can the Cellometer software count platelets? | Yes, the Cellometer Auto 2000, Cellometer K2, Cellometer X2 and Cellometer Spectrum 10x can count platelets when stained with calcein AM. |
| Can the Cellometer software count primary cells and cell lines? | Yes. We offer a range of instruments, optimized for specific cell types. We have brightfield instruments for cell lines and purified primary samples; and we have fluorescent instruments for primary, messy cell types. Hundreds of cell types can be counted on our instruments and the list continues to grow. |
| Can the Cellometer software count stem cells? | Yes. For stem cells, we recommend either the Cellometer Auto 2000, Cellometer K2 or the Cellometer Spectrum. These are dual fluorescent systems optimized for primary cell samples such as stem cells. |
| Can the Cellometer software count yeast? | Yes. The Cellometer X2 is optimized with higher magnification to count yeast cells, which are typically smaller than 5 microns. |
| Can the Cellometer software detect and count cells that are labeled with antibodies against surface markers? | Yes, for a surface marker, we recommend the Cellometer Spectrum. This system has cameras that are more sensitive, which optimizes the systems for these assays. |
| Can the Cellometer software measure heterogeneous cell populations? | Yes, the cells spread into a thin layer within the counting chamber. The same sample can be measured multiple times using different parameters. |
| Can you use glass slides in the Cellometer? | No, you can only use our disposable cell counting chambers in the Cellometer instruments. |
| Do any of the Cellometer instruments have the capabilities to have more than 2 filters? | No, all Cellometer systems have a maximum of 2 filter optics modules. However, with our Cellometer Spectrum instruments, the filters are interchangeable, and the user can therefore run more than just the S1-534-470 (Green) & S1-655-527 (Red) filter set. We offer the additional following filters to customize your Spectrum system: S1-452-3652 (Blue); S1-605-527 (Orange); S1-692-620 (Far-Red). |
| Do I need a computer to operate the Cellometer Instruments? | Yes. The instruments that don't need a computer are the Cellometer Ascend and Cellometer Auto 2000. All other Cellometer instruments connect to a PC via USB cable. An existing PC in your lab may be used, but if it is also running other instruments via USB, those instruments may cause communication issues with the Cellometer camera drivers and Cellometer software. Be sure to disconnect other USB instrumentation before using the Cellometer. A laptop PC with factory-loaded software is also available for purchase with your Cellometer instrument. |
| Do I need to uninstall my old Cellometer software in order to upgrade? | No, there is never a need to uninstall your old software. The upgrade will overwrite the existing software to reflect the newest version. |
| Do we need to calibrate our Cellometer instrument before using it? | No, the Cellometer was designed to be a low maintenance instrument. There is no calibration that needs to be done. Required maintenance is similar to a microscope. If you wish, we do offer IQOQ and Preventative Maintenance services, to have your instrument tested. We also offer beads at a known concentration if you need to document the performance of your instrument periodically. |
| Does each Cellometer count trypan blue positive and negative cells at the same time, in the same sample? | Yes, if Trypan blue-stained cells are loaded into the counting chamber and the “Test Viability” option is selected, both Trypan Blue positive and negative cells will be measured at the same time, from the same sample. |
| Does each Cellometer instrument measure cell size? | Yes. Cell size is measured from the image. Mean cell diameter is included in the cell counting results. Diameters of all counted cells and a cell size histogram are included in “Analysis” function. |
| Does the Cellometer instrument automatically mix my cell sample with trypan blue? | No, the Trypan blue mixing is performed by the user prior to loading the disposable counting chamber. This is the same procedure that is used to count cells with a manual hemacytometer. |
| How do I get started using the Cellometer K2, X1, or X2? | We recommend reading through the Quick Start Guide that is shipped with your instrument. If you are looking for more assistance, please contact our Support Team via cellc-support@revvity.com |
| How do I get started using the Cellometer Mini? | We recommend reading through the Quick Start Guide that is shipped with your instrument. If you are looking for more assistance, please contact our Support Team via cellc-support@revvity.com |
| How do I get started using the Cellometer Spectrum? | We recommend reading through the Quick Start Guide that is shipped with your instrument. If you are looking for more assistance, please contact our Support Team via cellc-support@revvity.com |
| How do I store one of my Cellometer assays to its default settings? | To obtain the original assay settings, simply delete the changed assay and import the original assay. You can do this by clicking Options, then Import/Export assay, then highlighting the original assay and select import highlighted. If you want to keep your changed assay, click the pencil next to the assay in order to edit it. Then check ‘Save as a new assay’ and rename your assay. Once done, click “Save”. |
| How do we mitigate when cells are layered so the Cellometer seems to focus on a layer, but we can clearly see cells blurry in the background that look like a lower layer? | To mitigate the layered effect you are seeing, let the sample settle before counting. We recommend loading the slide and preview the brightfield image until you don’t observe any movement. |
| How fast can I count one sample using the Cellometer instrument? | Counting time depends on the number of cells and morphology of the sample, as well as the computer speed. Typically, cell lines require less than 30 seconds per sample. |
| How long does the LED light source last in the Cellometer instrument? | The light source is a light-emitting diode which has a very long lifetime and is estimated to last for the life of the instrument. |
| How much of my sample do I load into my Cellometer slides? | PD100, SD100 and ASD-CHM3 slides require 20 µl of sample. SD025 slides require 4 µl of sample and PD300 slides require 58 µl of sample, ASD-CHM8 require 10 µl of sample. |
| I would like to validate that my Cellometer is functioning properly by utilizing the Check Validation Bead Solution (CCBM). Which values should I pay attention to on the Certificate of Analysis, and what do they refer to? | The Certificate of Analysis (CoA) contains a lot of useful information. Most of the values are ranges as opposed to discrete values. You’ll want to ensure that your results for the bead check fall inside the ranges listed for “Concentration” and “Specification: % Green FL Beads”. The “Concentration” range refers to the overall concentration of the beads, whereas the “Specification: % Green FL Beads” refers to the concentration of green fluorescent beads. These represent the “live” population of cells. |
| Is there a way I can upgrade my Cellometer software online? | Please contact Revvity support team at cellc-support@revvity.com to discuss upgrade procedures. |
| There are bubbles in my Cellometer chamber. What is causing this and how can I avoid it? | These bubbles are usually caused by vortexing/shaking the sample prior to loading. You may be pipetting vigorously during the mixing process - this could also cause bubbles prior to sample loading. |
| What cell types have been analyzed using Cellometer? | Cellometer systems have been used to image and analyze a wide variety of sample types, including both primary cells and mixed cell populations. Optimized protocols for analysis of PMBCs, stem cells, primary hepatocytes, adipocytes, yeast, platelets, algae, and other sample types are available. |
| What does a good focus look like on the Cellometer? | Compared to looking for cellular architecture, on for example, a confocal microscope, the focus method is different on a Cellometer. In the case of the Cellometer, you want to see a crisp, dark membrane, and a bright white center for each cell. |
| What filters does my Cellometer instrument have? | Instrument User Manuals list the filters installed in your Cellometer system. |
| What fluorophores can I detect on the Cellometer Spectrum? | To figure out whether a fluorescence optic module (FOM) can detect certain fluorophores, please visit the Cellometer Spectrum web page. |
| What is the main difference between image-based cytometers (like a Cellometer) and flow cytometers? | Image-based cytometry allows you to image, analyze cells, and gate out debris and smaller particles based on a visual representation and in a flow-free system. |
| What is the target concentration range for the Cellometer instruments? | To find the concentration range of your Cellometer, along with other capabilities please visit our products page, select your instrument, and go to the “Spec” tab. |
| Where can I find the Cellometer manuals? | From within the Cellometer software: For Cellometer X2, Cellometer K2, Cellometer Auto T4: from the software screen, click the question mark in the lower-right corner, then click the first ‘Go’. The new window that loads is the manual. For the Cellometer Auto 2000, click the “?” on the bottom right of the assay home screen and the manual will open up. Manuals can also be found via the Revvity instrument web page. |
| Why is it important to unplug both the USB and power cord from the instrument when upgrading Cellometer software? | The reason is that drivers can remain within the computer, the instrument, or both when upgrading. If you leave the USB connected, you will get an error saying the software cannot be installed. If the power cord isn’t unplugged from the instrument, old drivers stay active in the camera and the new drivers will fail to install. |
| Why is it important to use the correct power cords for the Cellometer instrument? | Cellometer instruments have specific power cords with respect to the voltage supplied to the instrument. Using the incorrect power cords can result in internal damage or lack of functioning mechanical parts. |
Cellometer Auto 2000
| Question | Answer |
|---|---|
| Can I connect a printer to work with my Cellometer Auto 2000? | Yes. Printers work on the Auto 2000 and we recommend using an up-to-date printer. Older printers can have driver or hardware issues that will not always work on the Cellometer Auto 2000. It is recommended to work with your IT department to help decide which printer to use. |
| Can you save data to the hard drive on the Cellometer Auto 2000? | Yes, Cellometer Auto 2000 has onboard storage. |
| Does my Cellometer Auto 2000 have onboard storage? | Yes, the Cellometer Auto 2000 has a built-in hard drive which serves as onboard storage. |
| How do I get started using the Cellometer Auto 2000? | We recommend reading through the Quick Start Guide that is shipped with your instrument. If you are looking for more assistance, please contact our Support Team via cellc-support@revvity.com |
| I’m getting an error on my Auto 2000 display that states, “Operating system not found”. How should I proceed? | Please contact cellc-support@revvity.com to coordinate the repair of your instrument. |
| It seems the software on the Cellometer Auto 2000 has frozen after counting and I cannot click anything. | Check to see if "Auto-Save Images" is activated by going to Settings > Saving Options. The more images and data the software is saving, (i.e. AO/PI assay, Raw AND counted images, etc.) the longer it takes to save to a USB or network drive. If you do not need to save images but have the option activated, you can turn it off. If that does not work, try connecting a mouse to see if you can click on the software. |
| My Cellometer Auto 2000 or Cellometer Auto 1000 screen is frozen! Is there anything I can do? | First try plugging in a mouse and keyboard (if needed) to see if you can run your samples. Please contact cellc-support@revvity.com about this issue. |
| When I click on my Auto 1000/Auto 2000 touchscreen, the mouse arrow is not corresponding with the location I am touching. How can I fix this? | 1. Use the mouse to hover over the bottom left of the screen to open the start menu. 2. Select “DMC Touch Panel Configuration” 3. When the screen pops up, click “Calibration” on the bottom left corner. 4. Use the pipette tip to click the corner of the blue corners that pop up on the screen. 5. Click “OK” |
| When I open up my Cellometer Auto 2000, it tells me that it is running in “data analysis mode”. How can I fix this? | First, make sure that you only have one window of the software open. Next, try a hard reset of your Auto 2000 (unplug the instrument, remove any USBs connected, and wait 5-10 minutes before plugging it back in and trying to power on the instrument again). If this does not work, please contact cellc-support@revvity.com |
Cellometer Auto T4
| Question | Answer |
|---|---|
| How do I get started using the Cellometer Auto T4? | We recommend reading through the Quick Start Guide that is shipped with your instrument. If you are looking for more assistance, please contact our Support Team via cellc-support@revvity.com |
| Why do you suggest a 0.1% trypan blue final concentration on the Cellometer Auto T4? | Any higher concentration (0.2% and greater) can cause cell viability to decrease quicker, as a result of the cytotoxicity of the dye. Also, higher concentrations of Trypan blue causes a darker background image that can give viable cells the appearance of dead cells. 0.1% Trypan blue will still stain the dead cells very clearly. |
Error messages
| Question | Answer |
|---|---|
| An error is coming up every time I click “Preview Image” and it says: “Error Setting up Preview”. | There could be something wrong with the USB communication between the computer and the camera. Try changing USB ports on the computer. Over time USB ports tend to lose connectivity. |
| What does the error “Could not read GPIO values” mean? | The GPIO value error displayed indicates the computer has lost communication with the camera. This error commonly occurs when the Cellometer is shut down without first closing out of the Cellometer software. |
Miscellaneous
| Question | Answer |
|---|---|
| How do I know if I have the correct fluorescent filters for a particular fluorophore? | Consult your manual for the current filters installed in your system. Use the excitation/emission values to verify your fluorophore is detectable using an online spectral viewer. For more information use https://fluorofinder.com/ |
| How do I know if my instrument counting correctly? | Our cell counting and image cytometry instruments have built-in functions that allow for visual quality control. In Cellometer and Cellaca systems, this function is called “Counted”. After counting an image, check the “Counted” box. Toggle between the counted and the raw image to verify all your cells are being counted correctly. In the Celigo software, turn on the graphic overlay. If you have questions, please contact cellc-support@revvity.com |
| How do I run my first sample? | We recommend reading through the Quick Start Guide that is shipped with your instrument. If you are looking for more assistance, please contact our Support Team via cellc-support@revvity.com |
| Should I turn off the instrument daily/or after each use? | There is no requirement for turning the instrument off, but we would recommend turning it off once a week, for example, over the weekend. |
| What are the emission and excitations of the VB-535-402 and VB-660-503 filters? | The VB-535-402 has an excitation of 470 nm and an emission of 535 nm. The VB-660-503 filter has an excitation of 540 nm and an emission of 660 nm. |
| What is the acceptable amount of variation between counts? | In general, variation should be less than or equal to 10%. |
| What is the password for the laptop that comes with my instrument? | The laptops do not have passwords. The usernames are simply Cellometer (for Cellometer line instruments), Celigo, and Cellaca. |
Ordering/Purchasing
| Question | Answer |
|---|---|
| How do I order consumables and reagents? | You can order slides via www.revvity.com/shop |
| We had to get a new laptop for our lab. Is it possible to purchase the Cellometer software without buying a new instrument? | Yes. There is no charge to current users for the software if the Cellometer has been purchased through Revvity and/or its affiliated companies or an authorized distributor. Please contact cellc-support@revvity.com regarding the installation procedure. |
Reagents
| Question | Answer |
|---|---|
| Can Revvity cell counting instruments detect dual fluorescence stains, meaning a live and dead staining kit?If so, would this help when we are trying to count cells in a sample with high solid content? | Yes, our fluorescent instruments can detect dual fluorescence stains. We recommend our AO/PI staining solution for live and dead staining. The AO (Acridine Orange) enters all cells and fluoresces green upon binding to DNA. The PI (Propidium Iodide) only enters cells with compromised cell membranes (i.e. dead or dying cells). The PI will quench the AO signal and fluoresce red, so you will clearly be able to see which cells are alive and which are dead based on whether they are green or red. |
Sample preparation
| Question | Answer |
|---|---|
| Do I need any special buffer or reagent for cell counting? | No special buffer is required. Cells suspended in growth media or PBS are routinely counted. Trypan blue or AO/PI (part no. CS2-0106) is used for viability assays. For yeast counting and viability, the yeast cells need to be first diluted in a Yeast Dilution Buffer (available from Revvity) prior to staining with Yeast AO/PI (part no. CSK-0102). |
| How can I minimize CV in my yeast samples? | Try to have a high amount of yeast cells in the image. We suggest around 500 minimum. |
| How do I deal with clumpy cells? | There are a few ways to address clumpy samples. The first way is to make sure your sample has been adequately mixed using a pipette or a gentle vortex. After you’ve loaded the sample, make sure that your focus shows crisp, dark membranes, especially within the clusters. We suggest you first run a count and then visually QC the images using the “Counted” function. This will show you how the instrument is parsing the cells in these clusters. If you are concerned with the way that the software is de-clustering, please contact cellc-support@revvity.com. We can help you to analyze your sample to optimize the de-clustering parameters. |
| How do I determine the dilution factor? | The formula for dilution factor (DF) is as follows: DF = (final volume of cells + stain)/(initial volume of cells). For example, if you mix your sample 1:1 with AO/PI, you’ll need to add 20 µL AO/PI to 20 µL cells, for a total of 40 µL. So, DF = (40 µL)/(20µL cells) = 2. If you choose to dilute your cells prior to adding stains, you’ll also need to account for this in your DF. Use the same formula, except now it looks like this: DF (final volume of cells & diluent)/ (initial volume of cells). For example, if you dilute 20 µL of your cells in 40 µL of media, DF = (60µL)/(20µL) = 3. To account for both stain and sample dilution prior to staining in this case, you would multiply these two dilution factors to get a value of DF=6. |
| My yeast sample is dirty and has a lot of non-cellular debris. What is the best way to measure viability in this case? | For yeast samples that display a lot of non-cellular debris in brightfield, we recommend using a mixture of acridine orange and propidium iodide (AO/PI). However, you must dilute your cells with our yeast dilution buffer prior to staining with AO/PI. The buffer adjusts the pH of the sample to optimize live/dead staining of the yeast cells. |
| What would you say is the minimum pH a solution of cells needs to have in order for AO/PI to stain effectively? | A pH of 7. Acidic Solutions make the AO staining ineffective. We recommend diluting your sample in media or neutral buffer to allow for a more neutral staining environment. |
| When using PI as a stain, should we be diluting it and our samples with a 0.9% saline solution instead of sterile water?If so, why? | It is preferable to dilute with a saline solution for two reasons. The first reason is that it creates a pH neutral environment (optimal conditions for PI staining). The second reason is that water can have some lysing effects on the yeast. |
Slides
| Question | Answer |
|---|---|
| How many counts can you do with one of your slides? | With all Cellometer slides except Cellometer Ascend slides, you can perform two counts per slide. If you only use one chamber of the slide for a count, you can save the other chamber for later, just be sure to keep it clean and free of dust or fingerprints until ready to use. Cellometer Ascend slides can accommodate either 3 or 8 samples per slide. |
| What is the difference between the PD100 slides and SD100 slides? | The PD100 slides are 100% inspected and are covered by warranty for 24 months from the date of manufacture. The PD100 slides are individually slotted in a microscope slide box. The PD100 slides are ready to use right out of the microscope slide box. The SD100 slides are covered by warranty for 12 months from the date of manufacture. A percentage of the SD100 slides are inspected for quality. The SD100 slides are packaged in a cardboard box and there is a protective film on both sides of the slide that must be removed before use that protects against scratching. |
| What volume is used on a PD300? Do I need to change any software settings with these slides? | The volume to load for the PD300 slides is 58 µL. Yes, you will need to indicate this change in the assay parameters. It is important to do this so the concentration can be calculated properly by the software. Simply select the “Edit Assay” pencil on the lefthand side of the screen (or the “Edit Assay” button at the top of the Auto1000/2000 screen), turn “on” the option for “Special Cells,” and select the type of slide you are using. |
Software/Data analysis
| Question | Answer |
|---|---|
| Can I analyze the data on my computer? Is there an analysis software package I can install on my computer? | A satellite computer with Celigo software can be purchased separately. These are specific computers that support the high image processing demands for image analysis. |
| Can you image at different focal planes in the same scan? | With the Celigo system, yes, use 5-channel Target 1+2+3+4+5 with image offset. |
| How can I take a look at the data for an individual cell? | After running a count, close out of the results page to take a look at your images. On the right-hand side of the screen, check the “Counted” box. This will circle the cells that the Cellometer counted in green, and the objects not counted in yellow. You can click on these individual cells to open a dialogue box. This box includes information on diameter, circularity, area, as well as fluorescent intensity (for fluorescent instruments). |
| How can you quickly set up the exposure time of different channels in the scan tab on the Celigo system? | Setup the focus for the channel that has content in every well first, e.g. brightfield or Hoechst stained cells. Then switch to another channel to setup the focus offset, then use the pixels in the objects to determine if the exposure needs to be increased or decreased. The pixel range is 0 (black) to 255 (white). Ideally, object pixels should be between 50-90% of that range (135-250). Repeat this for each of the channels. Save this as an experiment/project for settings to be automatically applied in the next scan. |
| How do analysis parameters work, when to use them? | Analysis parameters tell the software how to count the raw image of cells. You can adjust these to gate out cells based on their morphological characteristics such as size, roundness, etc. You can also adjust the parameters to selectively count cells based on their fluorescent intensities. Perhaps you only want to count the cells that are between 10-20 microns and have a bright GFP signal. In a way, analysis parameters allow you to inform the software based on the biological properties of your sample. |
| How do I customize programs for different cell types? | Please contact cellc-support@revvity.com. Our team can create personalized cell types and assays to suit your specific experiment. |
| How do I set up parameters for a new cell type? | Please contact cellc-support@revvity.com. Our team can create personalized cell types and assays to suit your specific experiment. |
| How do I verify if my cells have been counted? | For direct cell counting assays, click the “counted” button after counting your sample. The counted cells will be outlined in green, allowing you to visually verify that your target cells have been counted. For fluorescent-based assays, click the “counted” button after counting your sample. The target cells will be outlined in green, and excluded cells will be outlined in red. Additionally, for viability assays, the live cells are outlined in green and the dead cells are outlined in red. |
| How does the Cellometer software count clumpy cells? | The Cellometer software recognizes the bright centers of viable cells within clumps. Clumps can be de-clustered to count individual cells or remain counted as one, depending on the user’s selection. |
| How does the Cellometer software count different size cells? | Through cell type parameters, the user defines the size range to be counted. Depending on the application, different-sized cells can be included or excluded. This allows the same cell image to easily be analyzed for different cell types. |
| I cannot display an image, why? | There could be a few issues. First, we suggest taking a new background image. Make sure the instrument is powered on and connected to the computer via USB cable. If that does not work, try another USB port. If this is unsuccessful, please contact cellc-support@revvity.com |
| I cannot see anything in the fluorescent channel images. They are completely black/green, what’s happening? | If you cannot see anything in the fluorescent channels, first check the Assay Editor to make sure the correct lamps/modules are selected. Make sure the exposure time is set appropriately. If you see images that are too bright, reduce the exposure time incrementally until the ideal setting is found. If you have a sample of the fluorescent beads (shipped with all fluorescent instruments), prepare a slide of the beads. This is useful for confirming the instrument channels are working appropriately. |
| I’ve loaded my sample, but the count button is greyed out, what is going on? | You may have the “two-chamber assay” option checked within your software. You will need to uncheck that box and the count button will no longer be greyed out. You can do this by editing your assay and unchecking the box underneath the imaging mode box. |
| Is the K2 software backward compatible? For example, can you recount images from 3.0.0.9 on 3.1.0.8? | Yes, it can read images from older version of the software. Be aware the counting algorithms have had improvements in the newer versions of the software, and recounting old results might result in slightly different results due to the improvements. |
| Using the Celigo image cytometer, what do you do when the segmentation results on ANALYZE Tab are different from the RESULT Tab? | When in Analyze tab, switch the well view to whole well view. This will display all the images of the well and on-the- fly analysis will look at the whole well, not just the FOV. This is more inline with how the data set is analyzed and displayed in the Results tab. |
| What are FOV and FOR and what are the differences? | Field of View (FOV) is the area of one image that the camera can capture. Field of Regard (FOR) on the Celigo system is the area the galvanometer mirrors and camera can image without moving the stage. |
| What are the computer requirements to run the Cellometer software? | The specification sheet for each Cellometer instrument provides the requirements needed to run the Cellometer software. Find the specification sheet via the Revvity web page for your instrument. |
| What does the Advanced BR/F mode do? | Advanced BR/F mode, when turned on, uses the brightfield image to help decluster the cells. This is a setting you turn on in Cell Type parameters under “Fluorescent (FL)” tab. |
| What is a “mask” and how do you use it? | A mask or single mask is when one channel of a multichannel scan is used to identify objects of interest. The area of this object is analyzed for pixel intensity in the other image channels where they may or may not have a fluorescent signal. For example, Hoechst-stained cells are identified as the mask, and the second channel is green looking for GFP expression within that cell. This provides a total count and GFP count, therefore % GFP expression can be calculated. |
| What is the area of the counting chamber analyzed? | The area the Cellometer images and analyzes is similar to the area of 8 quadrants on a standard hemacytometer. Image A is roughly equivalent to 2 quadrants, Image B is roughly equivalent to 2 quadrants, Image C and D etc. |
| What is the best way to focus on my sample? | When you are focusing the instrument, use the knob on the side of the instrument (or the focus arrows on Cellometer Auto 2000) to adjust the focus. Make sure your sample is settled, as the focus might change as the cells settle into the slide. Then, adjust the focus until you see a bright-white center and a crisp, dark cell membrane for your cells. |
| What is the difference between assay settings and cell type settings? | Assay settings pertain more to the particular stain you are using to count cells/measure viability/etc. In these settings, you will edit exposure times, imaging mode, and the cell type corresponding to that particular assay. Cell type settings, on the other hand, get into the fine details of your cell morphology. For example, you can edit the size cutoffs for your cells, adjust the roundness value for more oddly shaped cells. You can keep all other aspects of an assay the same and change the cell type. |
| What is the standard method of measuring viability in yeast cells? | We recommend using propidium iodide (PI) to stain yeast cells. |
| When I compare image A and image B, image B is darker. How can I fix this? | The first step is to try taking a new background image. If this does not solve the problem, the power supply may be faulty. Please contact cellc-support@revvity.com |
| When I try to take a background image, I get a message saying the dim count is very low. What does this mean? | This may mean that the LEDs need to be replaced in your instrument. Please contact cellc-support@revvity.com |
| Why aren’t all my cells being outlined as counted? | It may be that some cells are being excluded based on your parameters. The software may be excluding cells based on size, roundness, or FL intensity depending on the settings in your assay. To change these parameters, click the pencil button next to your cell type. |
| Why does my sample look blurry? | If you are using the SD100 slides, double-check to see that you have removed both strips of plastic from both sides of the slide. Leaving these on can create a strange, blurred effect in the image. |
| Why is proper focus important? | The proper focus (crisp dark membranes and bright white centers) helps the software to identify the cells, separate them from background noise, differentiate between live and dead cells, and decluster individual cells in a clump. |
| Will cell debris be counted? | No. In a typical cell culture, cell debris is smaller in size and different in morphology than live cells. The Cellometer software excludes debris because it will fall outside of the requirements to be counted as a cell. For fluorescent instruments, the debris will not be stained when using an enzymatic or nuclear dye. |
Support and training
| Question | Answer |
|---|---|
| How do I reach support or submit a support ticket? | Contact us via cellc-support@revvity.com or via www.revvity.com/contact-us |
| How do I request training? | Contact us via cellc-support@revvity.com or via www.revvity.com/contact-us |
Warranty
| Question | Answer |
|---|---|
| How do I find warranty information? | New Instrument Warranty Registration is from the first 12-months of the original ship date. Contact cellc-support@revvity.com for an extended warranty quote. |
| How long are your reagents under warranty? | Warranty is valid until the expiration date stated on the product label. |
| Where can I register my instrument to receive warranty benefits? | New instrument warranty is valid for 12 months from the date of product receipt. Contact cellc-support@revvity.com for an extended warranty quote. |
For research use only. Not for use in diagnostic procedures. The information provided above is for informational and research purposes only. Revvity assumes no liability for any injuries, losses, damages, or outcomes arising from the use or misuse of this information or its recommendations. This information is provided "as is" without warranties. Users are solely responsible for determining the suitability of recommendations for their research and should not substitute them for their own professional judgment.