A CD19-positive human B cell acute lymphoblastic leukemia cell line (NALM6 cells; Cedarlane) was utilized for this study. NALM6 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (Wisent) and 5 ml antibiotic–antimycotic (100 × ; Thermo Fisher). NALM6 cells were engineered to stably co-express the fluorescence reporter tdTomato (tdT) and a codon-optimized bioluminescence firefly luciferase reporter (Luc2) using a lentiviral vector previously constructed in our lab [21 ]. Cells were transduced with lentiviral vector using polybrene (1.6 µg/ml, Sigma Aldrich). Transduced cells were analyzed and sorted using fluorescence-activated cell sorting (FACSAria III flow cytometric cell sorter, BD Biosciences) and expanded prior to downstream use.
Facsaria 3 flow cytometric cell sorter
Manufactured by BD
Sourced in United States
The FACSAria III is a flow cytometric cell sorter. It is designed to sort and analyze cells based on their physical and fluorescent characteristics.
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Lab products found in correlation
5 protocols using facsaria 3 flow cytometric cell sorter
1
Generating Engineered NALM6 Cell Line
2
Bioluminescent 4T1BR5 Cell Line
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Brain-seeking mouse mammary carcinoma cells (4T1BR5) were a kind gift from Dr. Patricia Steeg's lab (NIH, Centre for Cancer Research) and engineered to stably coexpress red-shifted Luciola italica luciferase (FLuc) and GFP using a commercial lentiviral vector (RediFect Red-FLuc-GFP lentiviral particles; PerkinElmer, USA). Cells were transduced at a multiplicity of infection of 20 and sorted based on GFP expression using a FACSAria III flow cytometric cell sorter (BD Biosciences, San Jose, CA, USA). The resultant 4T1BR5-FLuc-GFP cells were maintained in DMEM containing 10% FBS at 37°C and 5% CO2. All in vitro experiments were performed in triplicate.
3
Cell Cycle Analysis of Breast Cancer Cells
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Breast cancer cells (naïve and engineered 4T1BR5) were cultured as stated above. Cells were centrifuged at 1000 rpm for 5 minutes. Cell pellets were then fixed with 500 μl of 70% ethanol for 30 minutes in 4°C, washed twice with phosphate-buffered saline (PBS), and centrifuged at 850 g. Cells were then treated with 50 μL of RNase (100 μg/mL). The mixture was kept in a water bath at 37°C for 30 minutes prior to staining with 200 μL of propidium iodide solution (50 μg/mL) and then analyzed by flow cytometry using a FACSAria III flow cytometric cell sorter (BD Biosciences, San Jose, CA, USA).
4
Labeling 4T1-BR5 Cells with Magnetic Beads
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The 4T1-BR5 cells were received from Dr. Patricia Steeg’s lab and engineered to stably co-express red-shifted Luciola Italica luciferase (Red-FLuc) and green fluorescent protein (GFP) using a commercial lentiviral vector (RediFect Red-FLuc-GFP; PerkinElmer, USA). Cells were transduced and FACS sorted based on GFP expression using a FACSAria III flow cytometric cell sorter (BD Biosciences). The resultant 4T1BR5-Red-FLuc/GFP cells were maintained in DMEM containing 10% FBS and 1% antibiotics, at 37 °C and 5% CO2. For iron labeling, 2 × 106 4T1BR5-Red-FLuc/GFP cells were plated, and 24 hours later were incubated with 25 μg/mL of micron-sized superparamagnetic iron oxide (MPIO) beads for an additional 24 hours (0.9 μm in diameter, 63% magnetite, conjugated with Flash Red; Bangs Laboratory, Fishers, IN, USA). Cells were washed three times with Hanks balanced salt solution (HBSS), collected and thoroughly washed three more times with HBSS to wash off residual unincorporated MPIO before in vitro evaluation or injection into animals.
5
Establishing Dual-Labeled Breast Cancer Cells
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The 4T1BR5 cells were a kind gift from Dr. Patricia Steeg's lab and transduced with a commercial lentiviral vector (RediFect Red-FLuc-GFP; PerkinElmer, USA). Cells were FACS sorted based on GFP expression using a FACSAria III flow cytometric cell sorter (BD Biosciences, USA). The parental 4T1 cells were also received from Dr. Patricia Steeg's lab and transduced with an in-house RLuc8/ZsG lentivirus. Cells were sorted based on ZsG expression using FACS. The resultant 4T1BR5-FLuc/GFP (4T1BR5-Fluc) and 4T1-RLuc/ZsG (4T1-Rluc) cells were maintained in DMEM containing 10% FBS and 1% antibiotics, at 37°C and 5% CO2, and then transduced a second time using an in-house CD:UPRT/tdT lentivirus. Both cell lines were FACS sorted based on tdT expression. All lentiviral transductions were performed using a multiplicity of infection of 20 and in the presence of 8 μg/mL of polybrene. Cells were washed three times with Hanks balanced salt solution (HBSS) and collected for in vitro evaluation or injection into animals.
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