48–72 hours post transfection cells from each well of the 12-well plates were trypsinized with 0.1 mL 0.25% Trypsin-EDTA at 37°C for 3 min. Trypsin-EDTA was then neutralized by adding 0.9 mL of complete medium. The cell suspension was centrifuged at 1,000 rpm for 5 min and after removal of supernatants, the cell pellets were re-suspended in 0.5 mL PBS buffer. The cells were analyzed on a BD LSRFortessa flow analyzer. CFP was measured with a 445-nm laser and a 515/20 band-pass filter, and mKate with a 561-nm laser, 610 emission filter and 610/20 band-pass filter. For data analysis, 100,000 events were collected. A FSC (forward scatter)/SSC (side scatter) gate was generated using a un-transfected negative sample and applied to all cell samples. The mKate and CFP readings from un-transfected HEK293 cells were set as baseline values and were subtracted from all other experimental samples. The normalized mKate values (mKate/CFP) were then collected and processed by FlowJo. All experiments were performed in triplicates.
Lsrfortessa flow analyzer
Manufactured by BD
Sourced in United States
The LSRFortessa is a flow analyzer designed for high-performance cell analysis. It features advanced optics and a flexible configuration to deliver accurate and reliable data. The core function of the LSRFortessa is to analyze and quantify the phenotypic characteristics of cells in a sample.
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7 protocols using lsrfortessa flow analyzer
1
Flow Cytometry Analysis of Transfected Cells
2
Flow Cytometric Analysis of Transfected Cells
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A total of 48 h post-transfection, cells from each well of the 12-well plates were trypsinized with 250 µL 0.25% trypsin-EDTA at 37 °C for 5 min. Trypsin-EDTA was then neutralized by adding 750 µL of complete medium. The cell suspension was centrifuged at 1000 rotations per minute for 5 min, and after removal of supernatants, the cell pellets were resuspended in 0.5 mL phosphate-buffered saline buffer. The cells were analyzed on a BD Biosciences LSRFortessa flow analyzer (San Jose, CA, USA). YFP was measured with a 488 nm laser, a 535 nm emission filter and a 545/35 band-pass filter. For data analysis, 100,000 events were collected. A forward scatter/side scatter gate was generated using an un-transfected negative sample and applied to all cell samples. All experiments were performed in triplicates.
3
Flow Cytometry Analysis of Transfected Cells
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Forty-eight to 72 hours after transfection, cells from each well of the 12-well plates were trypsinized with 0.1 ml of 0.25% trypsin-EDTA at 37°C for 3 min. Trypsin-EDTA was then neutralized by adding 0.9 ml of complete medium. The cell suspension was centrifuged at 1000 rpm for 5 min, and, after removal of supernatants, the cell pellets were resuspended in 0.5 ml of PBS buffer. The cells were analyzed on a BD LSRFortessa flow analyzer. Cyan fluorescent protein (CFP) was measured with a 445-nm laser and a 515/20-nm band-pass filter, and mKate was measured with a 561-nm laser, 610-nm emission filter, and 610/20-nm band-pass filter. For data analysis, 100,000 events were collected. A forward scatter/side scatter gate was generated using an untransfected negative sample and applied to all cell samples. The mKate and CFP readings from untransfected HEK293 cells were set as baseline values and were subtracted from all other experimental samples. The normalized mKate values (mKate/CFP) were then collected and processed by FlowJo. All experiments were performed in triplicates.
4
Single-Cell Flow Cytometry of Harvested Cells
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Cells were harvested from chips by blocking one port of the basal channel with a tip and manually pipetting Cell Recovery Medium (Corning, 354253 200 µL chip−1) through the other port to push the ECM and cells out of the channel and harvest them. The harvested ECM was incubated in the Cell Recovery Medium for 1 h at 4 °C. The released single cells were centrifuged at 300 g for 5 min and resuspended in PBS for staining. Cells were first labeled with Live/Dead fixable dyes (1:1000 dilution; Invitrogen, L34963), followed by a 15 min incubation with fluorophore‐labeled antibodies and Fc Block (1:100 dilution) at 4 °C. Cells were washed twice and then fixed with Cytofix (BD Biosciences, 554655) for 15 min at RT. Cells were centrifuged and re‐suspended in PBS and stored at 4 °C prior to cytometric analysis using a using LSRFortessa flow analyzer (BD Biosciences). Results were analyzed using FlowJo V10 software (Flowjo, LLC) using a volume of 100 uL containing between 0.5 to 1 × 106 cells. Antibodies to CD19 (#340437), CD27 (#655429), CD3 (#641406), CD4 (#555346), and CD8 (#347314) were obtained from BD Biosciences; antibodies to IgD (#348210) and CXCR5 (#356919) were acquired from BioLegend.
5
Immune Response of Differentiated THP-1 Cells
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Differentiated THP-1 cells were stimulated with UI-MP or TBinf-MP at a ratio of 5 MP's per cell and cultured for 4, 24 and 48 h. Culture supernatants were harvested and cytokines measured by Cytometric bead array (BD Biosciences) according to the manufacturer's instructions using an LSRFortessa flow analyzer (BD Biosciences) [7] . Cells were harvested in 1 mL Trisure reagent (Bioline, Australia) for RNA purification and RT-PCR.
6
Flow Cytometry Analysis of Cell Lines
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For each cell line, ∼150 000 cells were seeded into one well of a 12-well cell culture treated plastic plate. After 24 h, the cells were trypsinized using 0.1 ml of 0.25% trypsin–EDTA. 0.9 ml of supplemented DMEM were added to neutralize the 0.25% trypsin–EDTA. The cell suspension was centrifuged at 1000 rpm for 5 min. After removal of the supernatant, the cell pellets were re-suspended in 0.5 ml of Dulbecco's phosphate buffered saline (Mediatech, catalog #21-030-CM). The cells were analyzed on a BD LSRFortessa flow analyzer. CFP was measured with a 445-nm laser and a 470/20 band-pass filter and mKate2 with a 561-nm laser, 600 emission filter and a 610/20 band-pass filter. A gate to SSC-width/SSC-area gates were first used to exclude doublets. Then, a FSC (forward scatter)/SSC (side scatter) gate was generated to select for live cell populations. All experiments were performed in triplicates.
7
Cell Harvesting and Flow Cytometry Analysis
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Approximately 48 h post transfection cells from each well of the 12-well plates were trypsinized with 0.2 mL 0.25% Trypsin-EDTA at 37 °C for 3 min. Trypsin-EDTA was then neutralized by adding 0.7 mL of complete medium. The cell suspension was centrifuged at 4000 rpm for 1 min and after removal of supernatants, the cell pellets were resuspended in 0.5 mL PBS buffer (Dulbecco’s Phosphate Buffered Saline; Mediatech, catalog #21-030-CM). The cells were analyzed on a BD LSRFortessa flow analyzer. TagCFP was measured with a 445-nm laser and a 470/20 band-pass filter, mKate2 with a 561-nm laser, 600 emission filter and 610/20 band-pass filter, and YFP with a 488-nm laser, a 525 emission filter and 545/35 band-pass filter. For all experiments performed 100,000 events were collected. A FSC (forward scatter)/SSC (side scatter) gate was generated using a un-transfected negative sample and applied to all cell samples. We use a compensation matrix on our flow cytometry data to remove cross-talk observed between the three fluorescent proteins (Supp. Fig. 13 ). Data processing was performed in FlowJo 7.6.5. All experiments were performed in triplicates.
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