3 × 106 cells seeded on a 15-cm plate a day before FACS analysis. On the day of FACS analysis, cells were incubated with RPMI-1640 containing amide-15N-glutamine and 10% dialyzed FBS for 1 hour. During the last 20 minutes of the 15N-isotopical labeling period, Hoechst 33342 (Thermo Scientific; 62249) at 10 μM was supplemented to the labeling media for DNA staining. Hoechst 33342-stained genomic DNA confirmed through ZEISS Axio Vert A1 Bio inverted microscope (ZEISS) coupled with PhotoLuoro LM-75 (89 North). Cells were trysinized and resuspended in identical, but cold (4 °C) labeling media. Cells were subjected to FACS analysis, BD FACSAria IIu SORP cell sorter (BD Biosciences).
Facsaria iiu sorp cell sorter
Manufactured by BD
The FACSAria IIu SORP cell sorter is a high-performance flow cytometry instrument designed for advanced cell sorting applications. Its core function is to rapidly analyze and precisely sort heterogeneous cell populations based on their physical and biological characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Blasticidin, by InvivoGen (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Zeiss (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin glutamine (psg), by Corning (1 mentions)
Dulbecco s modified eagle medium dmem, by Corning (1 mentions)
6 protocols using facsaria iiu sorp cell sorter
1
Isotopic Labeling and FACS Analysis
2
Cell Line Transduction and Characterization
3
Generating Stable NanoLuc Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A lentiviral vector containing NanoLuc engineered from Oplophorus gracilirostris (Nluc; ref. 45 (link)) was kindly provided by Dr. Preet Chaudhary (USC Keck School of Medicine). Nluc lentivirus was produced as described (35 (link)). To generate stable cell lines, 1 mL of viral supernatant with 10 μg/mL polybrene was added to 1 × 106 cells in a 6-well plate and cultured for 8 hours. Media were exchanged and 2 days later, cells were selected in 20 μg/mL blasticidin (InvivoGen). After selection, cell lines expressing NanoLuc were maintained in the media described above in the presence of 10 μg/mL blasticidin (InvivoGen).
To generate H2122 lacking HLAA3, H2122-Nluc cells (1 × 106) were transfected with 2 μg of the Cas9/single-guide RNA (sgRNA) vector PX458 (Addgene; plasmid 48138) using Lipofectamine 3000 (Thermo Fisher) in 6-well plates. The following oligonucleotides were used for cloning sgRNAs into pX458: HLAA3 forward, 5′-CACCGCATCCTGGATACTCACGACG-3′; HLAA3 reverse, 5′-aaacCGTCGTGAGTATCCAGGATGC-3′. Two days after transfection, GFP+ cells were purified by FACS using a FACSAria IIu SORP cell sorter (BD Biosciences), and single cells were seeded into a 96-well plate. Clones were screened for HLA-A*03 expression by flow-cytometric analysis (see below). These N-Luc cell lines were tested monthly for Mycoplasma infection.
To generate H2122 lacking HLAA3, H2122-Nluc cells (1 × 106) were transfected with 2 μg of the Cas9/single-guide RNA (sgRNA) vector PX458 (Addgene; plasmid 48138) using Lipofectamine 3000 (Thermo Fisher) in 6-well plates. The following oligonucleotides were used for cloning sgRNAs into pX458: HLAA3 forward, 5′-CACCGCATCCTGGATACTCACGACG-3′; HLAA3 reverse, 5′-aaacCGTCGTGAGTATCCAGGATGC-3′. Two days after transfection, GFP+ cells were purified by FACS using a FACSAria IIu SORP cell sorter (BD Biosciences), and single cells were seeded into a 96-well plate. Clones were screened for HLA-A*03 expression by flow-cytometric analysis (see below). These N-Luc cell lines were tested monthly for Mycoplasma infection.
4
Generation of Nanoluc-expressing Cell Lines
5
Flow Cytometric Isolation of EO Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EO cells were isolated and suspended in FACS buffer (1% FBS in PBS) as previously described (Chen et al., 1998 (link)). Fluorescent PE anti-rat CD90/mouse CD90.1 (Thy-1.1) (1:500; BioLegend) was used for 30 min at 4°C in the dark to allow to distinguish between fibroblasts and ameloblasts. The analysis was performed by a flow cytofluorimetry (FACSAria IIu SORP cell sorter; BD) using FACSDiva software. Data for 10 K to 20 K events were collected.
6
Isolation and Purification of PECAM1+ Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were trypsinized and stained by using 5 μl of anti‐human CD31 antibody (BioLegend, catalog no. 303110) diluted in 80 μl of phosphate‐buffered saline (PBS) with 20% fetal bovine serum (FBS) per 106 cells, for 1 hour at 4°C, after which cold PBS was used to wash the cells. The cell pellet was collected by centrifugation at 200g for 3 minutes. The cell pellet was resuspended in 350 µl of PBS with 20% FBS for sorting. PECAM1+ cells were sorted by using a FACSAria IIu SORP cell sorter (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com ) and collected in PBS containing 20% FBS.
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!