Image acquisition was preformed using Cellometer® Vision CBA Image Cytometer (Nexcelom Bioscience LLC.). For image analysis we used Nexcelom Data Package and MATLAB software. Nexcelom filter VB-595-502 was used for red fluorescence assay in Cellometer. We used Nexcelom image segmentation (Supplementary Figs. 3 and 8 ) for object detection upon setting Cell Diameter parameter to 2–5 μm for single cells and 5–20 μm for clumps95 (link). The Object Roundness parameter was set to 0.45-0.8 (with 1.0 used for perfectly circular shapes) to separate single living cells from debris.
Cellometer vision cba image cytometer
Manufactured by Revvity
The Cellometer® Vision CBA Image Cytometer is a compact, automated cell analysis system that utilizes image-based cytometry technology to count and analyze a variety of cell types. It provides rapid and accurate cell counting, cell viability assessment, and cell characterization in a user-friendly platform.
Automatically generated - may contain errors
4 protocols using cellometer vision cba image cytometer
1
Image-based Single-Cell Analysis Protocol
2
High-Frequency Irreversible Electroporation of 4T1 Mammary Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The murine 4T1 mammary tumor cell line was acquired from ATCC (Cat#CRL-2539). Cells were sub-cultured to approximately 80% confluence. All experiments were performed within the first 4–8 sub-cultures. At confluence, 4T1 cells were washed and resuspended in a 5.5:1 ratio of low-conductivity sucrose solution (85 g sucrose, 3.0 g glucose, 7.25 ml RPMI, and 992.75 ml DI water) to unsupplemented RPMI to 4 × 106 cells/ml. An 800 μl cell suspension was added to a 4 mm sterile electroporation cuvette (Fisher Scientific) and H-FIRE was delivered at a voltage of 204 V, 800 V, or 1600 V totaling 500, 2000, or 4000 V/cm electric field magnitude, respectively. Temperature was monitored by inserting a fiber optic temperature probe (Lumasense, Inc.) within the cuvette during pulsing. Shortly after pulsing ceased, the temperature probe was removed and the cells were maintained on ice. Aliquots were immediately removed for staining with acridine orange (AO)/propidium iodide (PI) for automated cell viability counting using a Cellometer Vision CBA Image Cytometer (Nexcelom) and trypan blue for manual cell viability counting via hemocytometer. Remaining cells were divided and plated at a density of approximately 600,000 cells/ml, and maintained in incubators for either 2, 8, or 24 h before supernatant was isolated for LDH Cytotoxicity Assay (Pierce) following manufacturer's protocol.
3
Quantifying Mitochondrial Content by Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cytometric analyses of mitochondria content, cells were immunostained in suspension with antibodies to mitochondrial proteins, including MTCO2 and TOM20. Briefly, cells were fixed in 4% formaldehyde at 37 ℃ for 10 min and permeabilized in a final concentration of 90% methanol. The fixed cells were washed twice with blocking buffer (0.5% BSA in PBS) by centrifugation at 3000 x g for 5 min and incubated with the indicated primary antibody in blocking buffer for 1 h at room temperature. Cells were washed twice with blocking buffer and stained with Alexa Flour® 488 or 647-conjugated goat anti-mouse IgG antibody in blocking buffer. Image and flow cytometric data of the immunostained cells were acquired using Cellometer Vision CBA Image Cytometer (Nexcelom) and FACSCalibur (BD Biosciences), respectively. FCS Express 6 Flow software (De Novo Software) was used for data analysis.
4
Piecewise Linear Fitting of Growth Curves
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To define the growth phases, we applied piecewise linear fitting using MATLAB. After plotting the absorbance (OD600)-based growth curve on a semilogarithmic scale, the algorithm defines the breakpoints in the behavior each growth curve. Based on the assigned breakpoint coordinates, we can further estimate the duration and the slope of each phase, defined by the two neighboring breakpoints. The AUC (Figs. 2 e, 6d , Supplementary Fig. 22 ) was calculated in MATLAB via trapezoid integration with unit spacing over the 71-hour period with 1-hour intervals. Cell count estimation from OD600 values (Supplementary Note 1 ) was based on OD600 measurements of twofold serial dilutions for each strain in liquid media with the Tecan Infinite® 200 PRO microplate reader, followed by imaging and cell counting of the same samples in the Cellometer® Vision CBA Image Cytometer (Nexcelom Bioscience LLC.).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!