Flow cytometry was performed to characterize cell populations present in samples collected during the apheresis process. A combination of stains and antibodies were used to identify cells containing DNA/RNA (SYBR Green I), white blood cells (WBCs) (CD45 antibody) and/or reticulocytes (CD71 antibody). Samples from subject 1 were stained with SYBR Green I (Molecular Probes); samples from subjects 2 and 3 were stained with SYBR Green I and CD45-PacificBlue; and samples from subject 4 were stained with SYBR Green I, CD45-Pacific Blue and/or CD71-APC. Samples were kept on ice or at 4–8 °C until analysed by flow cytometry.
Sybr green 1
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan, United Kingdom, Switzerland, China, Australia, Canada, Belgium, France
SYBR Green I is a fluorescent dye used in molecular biology for the detection and quantification of DNA. It binds to double-stranded DNA and emits a strong fluorescent signal upon excitation, allowing for the sensitive and specific measurement of DNA levels in various applications such as real-time PCR and DNA gel electrophoresis.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (57 mentions)
Trizol, by Thermo Fisher Scientific (33 mentions)
Rneasy mini kit, by Qiagen (19 mentions)
Facscalibur flow cytometer, by BD (18 mentions)
Accuri c6 flow cytometer, by BD (17 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (16 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (16 mentions)
Facscalibur, by BD (14 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (14 mentions)
Abi 7500ht system, by Thermo Fisher Scientific (12 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (11 mentions)
Accuri c6, by BD (11 mentions)
Propidium iodide, by Merck Group (11 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (10 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (10 mentions)
Primescript rt reagent kit, by Takara Bio (10 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (10 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (9 mentions)
M mlv reverse transcriptase, by Promega (8 mentions)
Premix ex taq, by Takara Bio (7 mentions)
788 protocols using sybr green 1
1
Characterizing Cell Populations by Flow Cytometry
2
Flow Cytometry for Microbial Enumeration
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Flow cytometry after nucleic acid staining was used to enumerate bacteria and viruses (14 (link), 18 , 27 (link)). Briefly, 1.5-mL samples were fixed with glutaraldehyde (0.5% final concentration), held at 4°C for 10–30 min, shock-frozen in liquid N2, and kept at −80°C until analyzed.
Immediately prior to the flow cytometry analysis, samples were thawed to room temperature and 0.5-mL subsamples were stained with SYBR Green I (Molecular Probes) in the dark for 10 min. Prokaryotes were enumerated on an Attune flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) by their signature in a plot of green fluorescence versus side scatter (14 (link), 18 ).
Viral abundance was also measured by flow cytometry after SYBR Green I staining (14 (link)). Prior to the analysis, samples were thawed and 0.5-mL subsamples were stained with SYBR Green I (Molecular Probes, Eugene, OR, USA) at a final concentration of 0.5× the manufacturer’s stock solution at 80°C for 10 min in the dark. Viruses were enumerated on the Attune flow cytometer as described above for prokaryotic abundance.
Immediately prior to the flow cytometry analysis, samples were thawed to room temperature and 0.5-mL subsamples were stained with SYBR Green I (Molecular Probes) in the dark for 10 min. Prokaryotes were enumerated on an Attune flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) by their signature in a plot of green fluorescence versus side scatter (14 (link), 18 ).
Viral abundance was also measured by flow cytometry after SYBR Green I staining (14 (link)). Prior to the analysis, samples were thawed and 0.5-mL subsamples were stained with SYBR Green I (Molecular Probes, Eugene, OR, USA) at a final concentration of 0.5× the manufacturer’s stock solution at 80°C for 10 min in the dark. Viruses were enumerated on the Attune flow cytometer as described above for prokaryotic abundance.
3
Growth Potential Evaluation of Cell Lines
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To examine growth potential, pooled 31T or SK-Mel-2 pooled clones were seeded into 96 well plates at 300 cells per well in either 1%, 2.5% or 10% serum-containing medium and incubated for 13–17 days. Samples were analyzed every 48 hr by lysing cells in 50 μl 0.2% SDS/well and incubating for 2 hour at 37°C prior to addition of 150 μl/well of SYBR Green I solution (1:750 SYBR Green I (Invitrogen-Molecular Probes-Carlsbad, CA) diluted in dH20). Plates were analyzed using a BMG Labtech FLOUstar Optima.
4
Quantification of Aquatic Microbiome
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Subsamples (2 ml) were fixed with glutaraldehyde (0.5% final concentration), incubated at 4 °C for 15 min in the dark, flash frozen in liquid nitrogen and then stored at − 80 °C [37 (link)]. With scatter diagrams of side scatter vs. red fluorescence and orange fluorescence vs. red fluorescence, the abundances of picoeukaryotes, Synechococcus and Prochlorococcus were directly determined by flow cytometry (Epics Altra II, Beckman Coulter, USA) [38 (link), 39 (link)]. The samples for heterotrophic bacterial counting were stained with 1.0 × 10−4 SYBR Green I (v/v, final concentration, Molecular Probes), incubated for 15 min in the dark and analysed by flow cytometry with scatter diagrams of side scatter vs. green fluorescence and red fluorescence vs. green fluorescence [38 (link), 39 (link)]. To obtain viral abundance, the samples were diluted with Tris–EDTA buffer (pH 8.0; Sigma), stained with 5.0 × 10−5 SYBR Green I (v/v, final concentration, Molecular Probes) and then incubated at 80 °C for 10 min in a thermostat water bath (DKB-501A, Shanghai Jinghong, China); abundance was then determined by flow cytometry [40 , 41 (link)]. Data analyses were performed with FCS Express V3 software (De Novo Software, http://www.denovosoftware.com/ ).
5
Melanoma Cell Growth Assay
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To examine cell growth, melanoma cell lines (501Mel, 55T and 108T) stably infected with either vector, WT RASA2, p.Arg310* mutant or p.Ser400Phe mutant RASA2 were seeded in 6 replicates into 96 well plates at 200-2000 cells per well and incubated for 7-17 days. Samples were analyzed every 48 hrs by lysing cells in 50 μl 0.2% SDS/well and incubating for 2 hour at 37°C prior to addition of 150 μl/well of SYBR Green I solution (1:750 SYBR Green I (Invitrogen-Molecular Probes) diluted in dH20).
6
Quantitative Real-Time PCR Analysis
7
Melanoma Cell Growth Assay
8
Cell Proliferation Assay with SYBR Green
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A375 and 74T cells were seeded into 96-well plates at 200 cells per well either in 2.5% or 10% serum-containing medium respectively and incubated for 7-10 days. Samples were analyzed every 24-48 hours by lysing cells in 50 μl 0.2% SDS/well and incubating for 2 hours at 37°C prior to addition of 150 μl/well of SYBR Green I solution (1:750 SYBR Green I (Invitrogen-Molecular Probes) diluted in dH20).
9
GRIN2A Knockdown Cell Growth
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To examine growth potential, stable pooled knockdown clones for GRIN2A were seeded into 96 well plates at 500 cells per well in either 1%, 2.5% or 10% serum-containing medium and incubated for 6–8 days. Samples were analyzed every 2–3 days by lysing cells in 50 μl 0.2% SDS/well and incubating for 2 hour at 37°C prior to addition of 150 μl/well of SYBR Green I solution (1:750 SYBR Green I (Invitrogen-Molecular Probes-Carlsbad, CA) diluted in dH20). Plates were analyzed using a BMG Labtech FLOUstar Optima.
10
Cell Growth Assay for A375 and colo829 Cells
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To examine cell growth, A375 and colo829 pooled RGS7 overexpressing clones were seeded in six replicates in 96-well plates, at 200–2,000 cells per well, and incubated for 7–17 days. Samples were analyzed every 48 h by lysing cells in 50 μl of 0.2% SDS/well and incubating for 2 h at 37 °C before the addition of 150 μl/well of SYBR Green I solution (1:750 dilution of SYBR Green I (Invitrogen-Molecular Probes) in distilled water).
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