Prior to viral transduction (24 h), 100,000 LentiX HEK293T cells were seeded into 6 well plates in DMEM. After 24 h, the media was replaced with DMEM containing a dilution series of matched MOIs of CMV-ZsGreen1-DR and CMV-mScarlet viral supernatants (2.5–200 μL), supplemented with 4 μg/mL polybrene (Millipore). After transduction (72 h), cells were harvested by trypsinization and reporter expression in each cell was quantitated on a Sony SH800Z flow cytometer, with 20,000 events captured in the FL-1 (ZsGreen1-DR) and FL-2 (mScarlet) channels. The percentage of positive cells for either reporter was then calculated based on an un-transfected control and viral titers (IFU/mL) with the following equation [(cells seeded × % positive cells)/mL viral supernatant].
Sh800z flow cytometer
Manufactured by Sony
The SH800Z flow cytometer is a laboratory instrument designed for cell analysis and sorting. It utilizes advanced optics and digital signal processing to precisely measure and analyze the physical and fluorescent characteristics of individual cells within a sample. The SH800Z is capable of rapidly processing and sorting cells based on user-defined parameters, making it a versatile tool for various applications in the field of cell biology and research.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated anti goat igg, by Thermo Fisher Scientific (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Fcs express 6, by De Novo Software (1 mentions)
5.0 μm pore size membrane filter, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Opti mem media, by Thermo Fisher Scientific (1 mentions)
Dm6000b microscope, by Leica (1 mentions)
8 protocols using sh800z flow cytometer
1
Quantifying Lentiviral Transduction Efficiency
2
Immunofluorescence and Flow Cytometry Analysis
3
GFP Expression Analysis by Flow Cytometry
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Cells were grown in synthetic complete media up to OD600 = 1 and washed with phosphate buffer saline. Resuspended cells were sonicated for two cycles of 30 s ON/10 s OFF with a Mandel Scientific ultrasonic sonicator at 50% output to disperse cell clusters. 488 nm laser of Sony SH800z flow cytometer was used to detect GFP, and LESH00SZFCPL™ software was used to generate the density plots and to analyze data. 100,000 events were screened for each strain, and GFP negative gate was set based on an isogenic strain lacking GFP. Data from at least three independent experiments were pooled in Microsoft Excel® to create %GFP negative graph and calculate standard deviation.
4
Flow Cytometric Analysis of Synchronized Phytoplankton
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Exponentially growing cultures were incubated for 48 h in the dark to synchronize cell division66 ,67 (link). Fifty mL of culture were filtered through a 5.0-μm pore size membrane filter (Millipore, Ireland), fixed with 1% paraformaldehyde for 10 min, washed in PBS (pH 7, Sigma-Aldrich, St. Louis, USA) and then centrifuged at 1200 g × 10 min. The pellet was resuspended in 2 mL of cold methanol and stored for at least 12 h at 4 °C to achieve chlorophyll extraction. The cells were then washed twice in PBS and the pellet was resuspended in 300 μL of propidium iodide (60 μg mL−1) and 30 μL of RNaseA (100 mg mL−1) for at least 2 h before analysis using a Sony SH800Z flow cytometer with a laser emitting at 488 nm. Duplicate samples were run at low speed and data were acquired in linear and log modes until at least 10000 events had been recorded. After aggregates and dividing cells (2 C) were discarded, gated Karenia vegetative populations (1 C) accounted for > 8000 events. The size of the smallest genome (K. mikimotoi) was calculated based on the size of the genomes of Alexandrium minutum strains VGO577 and AMP468 . The fluorescence emission of propidium iodide was detected at 620 nm. FCS Express 6 (De Novo Software, USA) was used to compute peak numbers, coefficients of variation (CVs) and peak ratios for the DNA fluorescence distributions in a population.
5
Lenti-GFP Transduction and CART Surface Expression
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Example 3
Cells transduced with Lenti-GFP as explained above were analysed on a Sony SH800Z flow cytometer with 488 laser. Signal from GFP transduced cells was compared with untransduced cells. The results are shown in
Constructs expressing irrelevant VH with a HIS tag were shown by flow cytometry to have surface expression on Jurkat cells using anti-His detection agents. This shows that the leader sequence directs the CART to the surface of the cell as expected.
6
CRISPR-Cas9 Transfection and Cell Sorting
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All experiments were performed in Human embryonic kidney 293 T (HEK293T) cells gifted by Ronny Drapkin. Cells were cultured in DMEM supplemented with L-glutamine (Thermo Fisher Scientific) and 10% FBS and incubated in a humidified incubator at 37 °C and 5% CO2. Plasmids and U6-sgRNA cassettes were transfected into HEK293T cells in 6 well plates using Optifect™ Reagent (Thermo Fisher Scientific) according to manufacturer’s instructions when cells were ~40–60%. Typically one well per plasmid/sgRNA condition is used in an experiment. Total DNA transfected was limited to 3–3.5 ug for plasmids and 100 ng for each U6 sgRNA cassette (up to 600 ng total) with 13–14 ul of Optifect™ reagent. Reagent was mixed and transfected into OptiMEM media (Thermo Fisher Scientific) without FBS and incubated for ~4–6 hours to increase transfection efficiency before supplementing FBS to 5%. Cells were recovered ~48 ± 2 hrs after transfection and sorted using a Sony SH800Z flow cytometer. Propidium iodide (PI) viability stain was added (to 1 ug/ml concentration) before sorting and cells were sorted for PI negative (viable) and GFP + cells. Attempts are made to control for variations in transfection efficiencies by setting a minimum GFP fluorescent gates and keeping flow cytometer settings constant when possible.
7
Quantifying Cellular GFP Expression
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Cells were grown in SC media to OD 600 = 1.0, and then vigorously vortexed to disperse cell clusters. Images were acquired by Leica DM 6000B microscope with brightfield and green filter. Alternatively, cells were diluted in a 96-well tray Biochem. Cell Biol. Downloaded from cdnsciencepub.com by 34.173.223.223 on 08/30/24 For personal use only.
and incubated at 25 • C, and cell clusters were analysed using Diskovery™ spinning disk confocal microscope with 60× lens. Images were quantified and analysed using Volocity™ software as in Rowlands et al. 2019b . Briefly, the pixel values of at least 25 ROIs (region of interest) with no cells were measured and the average signal was deemed background. Next, pixel values from ROIs over 100 cells in three separate fields were measured. Cells with signals higher than 120% of the background were considered GFP + cells. At least three independent experiments were conducted with each strain.
For flow cytometry, the cells were dispersed by sonication and fixed in 70% ice-cold ethanol prior to analysis by a Sony SH800z flow cytometer. Data were collected by the LESH00SZFCPL™ software. Gates of GFP -cells were delineated using isogenic strains with no GFP reporters. At least three experiments were conducted with each strain.
Data from the above assays were imported in Excel to calculate average values, standard deviations, and p values.
and incubated at 25 • C, and cell clusters were analysed using Diskovery™ spinning disk confocal microscope with 60× lens. Images were quantified and analysed using Volocity™ software as in Rowlands et al. 2019b . Briefly, the pixel values of at least 25 ROIs (region of interest) with no cells were measured and the average signal was deemed background. Next, pixel values from ROIs over 100 cells in three separate fields were measured. Cells with signals higher than 120% of the background were considered GFP + cells. At least three independent experiments were conducted with each strain.
For flow cytometry, the cells were dispersed by sonication and fixed in 70% ice-cold ethanol prior to analysis by a Sony SH800z flow cytometer. Data were collected by the LESH00SZFCPL™ software. Gates of GFP -cells were delineated using isogenic strains with no GFP reporters. At least three experiments were conducted with each strain.
Data from the above assays were imported in Excel to calculate average values, standard deviations, and p values.
8
Quantifying Lentiviral Transduction Efficiency
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Prior to viral transduction (24 h), 100,000 LentiX HEK293T cells were seeded into 6 well plates in DMEM. After 24 h, the media was replaced with DMEM containing a dilution series of matched MOIs of CMV-ZsGreen1-DR and CMV-mScarlet viral supernatants (2.5–200 μL), supplemented with 4 μg/mL polybrene (Millipore). After transduction (72 h), cells were harvested by trypsinization and reporter expression in each cell was quantitated on a Sony SH800Z flow cytometer, with 20,000 events captured in the FL-1 (ZsGreen1-DR) and FL-2 (mScarlet) channels. The percentage of positive cells for either reporter was then calculated based on an un-transfected control and viral titers (IFU/mL) with the following equation [(cells seeded × % positive cells)/mL viral supernatant].
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