To determine the effect of KD on cell growth and proliferation, each sample was subjected to a two-color competitive cell growth (CCG) assay [24 (link), 32 (link)] as follows. K562-CRISPRi cells transduced with a non-targeting sgRNA—expressing green fluorescent protein (GFP)—were co-seeded at 2 × 105 cells/ml in the same well as cells transduced with sgRNAs targeting the indicated gene—expressing BFP—at 2 × 105 cells/ml in 2 ml RPMI-1640 growth medium (day 0). The percentage of GFP- and BFP-expressing cells was determined every 2–4 days by flow cytometry for 14 days using the Agilent NovoCyte flow cytometer (Agilent, Santa Clara, CA, USA). Relative cell growth and proliferation was calculated as the proportion of BFP-expressing cells normalized to that at day 0.
Agilent novocyte flow cytometer
Manufactured by Agilent Technologies
Sourced in United States
The Agilent NovoCyte flow cytometer is an analytical instrument designed for cell analysis and sorting. It utilizes laser-based technology to detect and measure various characteristics of cells suspended in a fluid stream.
Automatically generated - may contain errors
4 protocols using agilent novocyte flow cytometer
1
Competitive Cell Growth Assay for Knockout Validation
2
CD20 Expression Analysis in OCI-LY3 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For analysis of cell CD20 staining, OCI-LY3 cells (1 × 107 cells) were collected and incubated with rabbit IgG isotype control (AC042, 1:50, ABclonal) or CD20 rabbit mAb (A4893, 1:50, ABclonal) for 30 min at room temperature, followed by goat anti-rabbit pAb Alexa Fluor 647 (AS086, ABclonal) staining for 30 min at room temperature. Non-fluorescently stained OCI-LY3 cells were used as blank control. The expression levels of CD20 were detected with an Agilent NovoCyte flow cytometer (Agilent). Each experiment was conducted three times.
3
Detecting Tfh Cells and B Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Tfh cell detection, mononuclear cells were incubated with anti-rat-CXCR5 (Abcam, Cambridge, UK; ab254415) at 4°C for 30 min, followed by staining with goat anti-rabbit IgG-Alexa Fluor 488 (Abcam; ab150077), anti-rat-CD4-BV421 (BD Biosciences; 743088), and anti-rat-ICOS-PE (eBioscience, Thermo Fisher Scientific, Waltham, USA; 12994981) for 30 min at 4°C. For B cell subset phenotyping, the cells were first stimulated with Leukocyte Activation Cocktail (BD Bioscience) for 6 h, then stained with anti-rat-CD45R-PE-Cyanine7 (Invitrogen, Waltham, USA; 25046082), anti-rat-CD3-APC (Invitrogen; 17003082) and anti-rat-CD27-PE (Invitrogen; 12027182). After cell surface staining, the cells were fixed, permeabilized and stained with anti-rat-IgG-BV605 (BioLegend, San Diego, USA; 405430). Cells were analyzed with the Agilent NovoCyte flow cytometer, and the data were analyzed with Agilent software v. 1.5.6 (Agilent Technologies, Palo Alto, USA).
4
Apoptosis and Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded 24 hours before treatment. Annexin V was stained using FITC Annexin V Apoptosis Detection Kit (BD Biosciences, 556547). Cells were trypsinized, washed twice with PBS, and resuspended in 100 μL of Annexin V binding buffer. Cells were incubated in the dark for 15 minutes after adding 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI). Next, 400 μL of Annexin V binding buffer was added, and cells were passed through a filter to remove clumps. For cell-cycle analysis, treated cells were trypsinized, washed twice with PBS, and fixed using ice-cold 70% ethanol. Cells were washed twice with PBS and resuspended in 500 μL of FxCycle PI/RNase staining solution (Invitrogen, F10797) and incubated at room temperature for 30 minutes. All flow cytometry experiments were performed on Agilent NovoCyte flow cytometer (Agilent) and analyzed using FlowJo v10.8.1 or Agilent NovoExpress v1.5.6.
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!