Binding of AlexaFluor488 Hu5F9-G4, BV786 mouse anti-human CD47 (Clone B6H12; BD Biosciences, San Jose, California, USA), or PE anti-mouse CD47 (Clone MIAP301; Biolegend, San Diego, California, USA) was assessed using an LSRII flow cytometer, and geometric mean fluorescence intensity was determined using FlowJo. To analyse the blocking ability of CV1-hIgG4, 1 × 106 cells were incubated with varying concentrations of CV1-hIgG4 for 15 minutes on ice. Cells were subsequently labelled using AlexaFluor488 Hu5F9-G4. Analysis was performed as described above, and data were fit to sigmoidal dose-response curves using Prism 6.
Lsrii flow cytometer
Manufactured by FlowJo
Sourced in United States
The LSRII flow cytometer is a high-performance analytical instrument used for the detection, identification, and quantification of cells, particles, and molecules in a sample. It utilizes the principles of flow cytometry to provide researchers with a powerful tool for multi-parameter analysis of complex samples. The LSRII is capable of simultaneously detecting multiple fluorescent and scatter signals from individual particles or cells, enabling the characterization of various cellular properties and functionalities.
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Lab products found in correlation
Cd3 e450, by Thermo Fisher Scientific (3 mentions)
Brefeldin a, by BioLegend (3 mentions)
Brefeldin a, by BD (2 mentions)
V bottom 96 well plate, by Corning (2 mentions)
Cytofix cytoperm buffer, by BD (2 mentions)
Anti cd11b, by Thermo Fisher Scientific (2 mentions)
Cd11c n418, by BioLegend (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
Mab7089, by R&D Systems (2 mentions)
Anti rat igg biotin f ab 2, by Jackson ImmunoResearch (2 mentions)
Efluor 506, by Thermo Fisher Scientific (2 mentions)
Cd4 pe cy7, by BioLegend (2 mentions)
Cd19 apc cy7, by BioLegend (2 mentions)
Cd8b precp cy5, by BioLegend (2 mentions)
Cd62l pacific blue, by BioLegend (2 mentions)
Klrg1 fitc, by BioLegend (2 mentions)
Cd127 alexa700, by Thermo Fisher Scientific (2 mentions)
Cd44 pe, by BioLegend (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Cd45 buv395, by BD (2 mentions)
99 protocols using lsrii flow cytometer
1
Quantifying CD47 Binding and Blocking
2
Multiparameter Tumor Immune Profiling
3
Mitochondrial Redox Species Assessment
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Mitochondrial-specific assessment of specific redox species was performed as previously described (Case et al., 2016 (link)). Briefly, cells were stained with 1 μM MitoSOX Red ( -sensitive mitochondrial-localized probe, Thermo Fisher Scientific #M36008, Waltham, MA, USA) for 30 min at 37°C. Cells were analyzed on a LSRII flow cytometer at 488/610 nm ex/em, and data analyzed using FlowJo software.
4
Multiparameter Flow Cytometry Analysis
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Cells were labelled with appropriate antibodies and sorted using sorter FASCAria (Becton Dickinson). Phycoerythrin (PE)-conjugated anti-mouse-CD11c (clone N418), -CD86 (clone GL1), -CD80 (clone 16-10A1), -CD31 (clone 390), -CD40 (clone 1c10), -major histocompatibility complexII (clone I-A), -CD115 (clone AFS98), -F4/80 (clone BM8) (from eBioscience). Anti-mouse PE–Cy7–CD8a (clone 53-6.7), Alexa Flour 700-CD4 (clone GK1.5), APC–Cy7–CD3ɛ (clone 145-2C11), PerCP–Cy 5.5–Gr-1 (Ly-6G/Ly-6C, clone RB6-8C5), APC–CD11b (clone M1/70), PE/Cy7–CD45 (clone 30-F11), FITC–Ly-6G (clone RB6-8C5) were purchased from PharMingen or Biolegend, PerCP/Cy5.5–CD16/32 (clone 93), Pacific Blue Ms CD3/Ly-6G(Ly-6C)/CD11b/CD45R(B220)/Ter-119, PE/Cy7–CD117 (clone 2B8), APC–CD115 (clone AfS98), PE–CD127 (clone A7R34), APC–Cy7–Ly6A/E (clone D7), FITC–CD34 (clone RAM34), APC–CD4 (clone RM4-5), FITC–CD44 (IM7), PerCP/Cy5.5–CD62L (MEL-14) were purchased from PharMingen, eBiocsience or Biolegend. Dead cells were discriminated using 4,6-diamidino-2-phenylindole. Samples were analysed on LSRII flow cytometer and the data files were analysed using FlowJo 5.5.5 software.
5
Comprehensive T-cell Immune Monitoring
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In vitro cultured PBMCs and TILs were incubated with 1 µg/mL of co-stimulatory antibodies αCD28/αCD49d (eBioscience) followed by stimulation in 96-well plates with complete medium, SEB (1 µg/mL) or total 5T4 peptide pool (5 µg/mL). Following a 2-hour incubation, brefeldin A and monensin (BD Bioscience) were added and cells were incubated overnight at 37°C. PBMCs were stained with surface antibodies, fixed/permeabilized and incubated with intracellular antibodies (online supplementary table S1 ). Dead cells were discriminated by live/dead fixable staining (Life Technologies). Samples were acquired on an LSRII flow cytometer and analyzed with FlowJo software.
Patients were deemed to be “responders” when the percentages of IFN-γ+ΤΝF-α+CD4+and CD8+ T cells after stimulation were higher than 0.02 and were at least 2-fold the background on analysis in at least one time point post-vaccination. In addition, for cultured PBMCs the delta values (5T4 value–background value) had to be ≥2-fold the corresponding pre-vaccination baseline delta value.
Patients were deemed to be “responders” when the percentages of IFN-γ+ΤΝF-α+CD4+and CD8+ T cells after stimulation were higher than 0.02 and were at least 2-fold the background on analysis in at least one time point post-vaccination. In addition, for cultured PBMCs the delta values (5T4 value–background value) had to be ≥2-fold the corresponding pre-vaccination baseline delta value.
6
Blocking CD47 Binding Assay
7
Evaluating Phenanthriplatin and TMV Uptake
8
Flow Cytometry and Mass Cytometry Analysis of SPCS1 Gene-Edited Jurkat T Cells
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The antibodies and conjugates used are listed in Supplemental Table 6 . For flow cytometry studies, WT and SPCS1 gene-edited Jurkat T cells were incubated with fluorophore-conjugated MAbs for 30 min at 4°C and then washed three times in PBS containing 5% FBS. Cells were immediately processed on an LSRII flow cytometer and data were analyzed using FlowJo 10.0.7. For mass cytometry studies, WT and SPCS1 gene-edited Jurkat T cells were labeled with MAbs conjugated with transition element isotopes and analyzed on a CyTOF 2 mass cytometer (Fluidigm DVS Sciences). Data were analyzed using Cytobank (http://wustl.cytobank.org ) and FlowJo 10.0.7.
9
Isolation and Phenotyping of Mouse Monocytes
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At each time point, mice were placed in a “hot box” and left at 38°C for 10 min. They were then placed in a restrainer, and the lateral tail vein was punctured using a 0.5 M EDTA (pH 7.4)-soaked 21-gauge needle. A single drop of blood was transferred to a 2-mL tube, and 10 μL 0.5 M EDTA was added to prevent clotting. Each sample was then mixed with 400 μL ice-cold PBS, placed on 300 μL Histopaque 1083 (Sigma-Aldrich), and spun at 400 × g for 30 min in a microcentrifuge. The monocytic layer was aspirated using a pipette, mixed with 1 mL ice-cold PBS, pelleted, and resuspended in 200 μL flow cytometry buffer (PBS, 5% fetal bovine serum, 0.05% sodium azide), and 1 μL of the cocktail of conjugated antibodies was added (1:200 dilution in each case). After 1 h incubation in the dark, cells were pelleted and resuspended in 2% paraformaldehyde in PBS, followed by a further 45-min incubation in the dark. The stained fixed cells were then pelleted, resuspended in filtered flow cytometry buffer, and transferred to standard flow cytometry tubes. Samples were analyzed using a BD Bioscience LSRII flow cytometer, with plots created and analyzed in FlowJo V.10.6.1. The following antibodies were used: CD45 (Thermo Fisher; 30-F11; Super Bright 600), CD3 (Thermo Fisher; 17A2; FITC [fluorescein isothiocyanate]), CD4 (Thermo Fisher; RM4-5; eFluor 450), and CD8 (Thermo Fisher; SK1; Alexa Fluor 780).
10
Characterizing Immune Cell Profiles
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Individual MLN from neonates and adults were harvested and placed in cold HBSS containing 1% calf serum (Gibco), 10 mM HEPES (Gibco), and 4 mM sodium azide. Cell suspensions were prepared by mincing tissues with scissors and pressing cells through wire mesh with 74 µm pore size (Compass Wire, Westville, NJ, USA). Cells were incubated in mouse Fc Block (CD16/CD32; BD Pharmingen, San Diego, CA, USA) for 5 min on ice, followed by a 30-min incubation with fluorochrome-conjugated antibodies specific for CD4, CD8, Ki67, or TCRαβ (BD Pharmingen). For intracellular cytokine staining, cells were activated with 50 ng/ml PMA and 0.5 µM ionomycin in the presence of 5 µg/ml brefeldin A, fixed and permeabilized, and stained with fluorochrome-conjugated anti-IFNγ (BD Pharmingen). Samples were run on a Becton Dickinson LSR II flow cytometer and analyzed with FlowJo flow cytometry analysis software.
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