Cell functionality was assessed by ICS, with Boolean gating to examine vaccine-induced HIV-1-specific CD4+ and CD8+ T-cell responses after stimulation with 3 different HIV 15-mer peptide pools (1 pool Gag, 1 pool Pol/Env, and 1 pool Nef peptides; JPT Peptide Technologies GmbH, Berlin, Germany). Staphylococcal enterotoxin B (SEB)-stimulated and unstimulated cells were used as positive and negative controls, respectively. The flow cytometry panel included a viability marker, CD3, CD4, and CD8 to determine T-cell lineage, and IFN-γ, TNF-α, and IL-2 antibodies. Data were acquired on a LSRFortessa 4-laser (488, 640, 561, and 405 nm) cytometer (BD Biosciences) and analyzed using FlowJo software version 9.9.4 (Tree Star, Inc.).
Lsr fortessa 4 laser
Manufactured by BD
Sourced in Belgium, United States
The BD LSR Fortessa 4 laser is a flow cytometer designed for multi-parameter analysis. It features four lasers, enabling detection of a wide range of fluorescent dyes and parameters. The instrument provides high-performance data acquisition and analysis capabilities for various applications in life science research.
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6 protocols using lsr fortessa 4 laser
1
Assessment of HIV-1-specific T-cell responses
2
Quantitative Immune Cell Analysis
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Cryopreserved PBMCs were thawed, filtered and stained with a B-cell or T-cell antibody cocktail for 30 minutes in PBS. Cells were washed with PBS and analyzed with a BD LSR Fortessa 4 laser cytometer. Cytometric analysis was performed using RUO FCS Express 7 (DeNovo Software).
3
Comprehensive Immune Cell Phenotyping
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Immune-cell phenotyping was performed using an LSR Fortessa 4-laser (488, 640, 561, and 405 nm) flow cytometer (BD Biosciences) and Diva software, version 6.2. FlowJo software, version 9.9.6 (Tree Star Inc.), was used for data analysis. CD4+ and CD8+ T cells were analyzed for CD45RA and CCR7 expression to identify the naive, memory, and effector cell subsets for coexpression of activation (HLA-DR and CD38) and exhaustion/senescence (CD57and PD1) markers. CD19+ B cell subsets were analyzed for the markers CD21 and CD27. ASC (plasmablasts) were identified as CD19+ cells expressing CD38 and CD27. We used CD16, CD56, and CD57 to identify NK cell subsets. γδ T cells were identified using an anti-TCR γδ antibody. HLA-DR, CD33, CD45RA, CD123, CD141, and CD1c were used to identify dendritic cell subsets, as previously described (See et al., 2017 (link)). Extracellular labeling was performed for all antibodies except for Ki 67 for which an intracellular labeling was performed with the BD cytofix/cytoperm kit (BD Biosciences).
4
Quantifying Phagocytic Activity in Peritoneal Exudate Cells
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Peritoneal exudate (PE) cells were isolated from sham-operated mice using ice-cold 30% sucrose solution in PBS. Sham mice, and not CLP mice were used because the latter condition is thought to induce phagocytosis before the phagocytosis assay is initiated and thus to interfere with the basal phagocytic phenotype of the peritoneal cells. E. coli bioparticles (Invitrogen, Merelbeke, Belgium) labeled with pHrodo fluorescent reagent were resuspended at 1 mg/mL. Bioparticles were added to the PE cells at a concentration of 1 × 10 6 cells/mL, as per manufacturer's instructions. All cells were incubated with bioparticles for 30 min at either 4°C (negative control) or 37°C and then processed using a fluoresscence activated cell sorting system (LSR Fortessa 4 laser; BD Biosciences, Erembodegem, Belgium) and analyzed using FlowJo software (Tree Star, Ashland, Ore) to determine the level of fluorescence. Percentage of PE-positive cells among CD45 + cells are shown in the figure. Cell suspensions were stained at 4°C in the dark with the following antibodies: life/death (APCCy7-efluor780) and anti-CD45 (BV510).
5
Comprehensive Flow Cytometry Analysis
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BM, spleen, and cells recovered from the ear dermis from infected and naïve mice were analyzed by flow cytometry. BM was harvested by flushing tibiae and femora in phosphate-buffered saline (PBS) solution and the cells were then passed through a 25-gauge needle to obtain a single cell suspension. Spleens were manually dissociated in PBS. Ear tissue homogenates, prepared as described above, were filtered in 5 mL round bottom polystyrene FACS tubes with 35 μm nylon mesh-screen filter before flow cytometry staining. PBS was supplemented with 0.1% bovine serum albumin (BSA) and 0.5 mm ethylene-diaminetetraacetic acid (EDTA) for flow cytometry staining, and non-specific staining was blocked using unlabeled anti-CD16/CD32 antibody (Fc block). Antibody combinations were modified from previously published studies, including by our group [16 (link),27 (link),28 (link),29 (link)]. Please see Table S1 for a complete list of antibodies. Cells were fixed with 2%PFA in PBS prior to acquisition of samples using a 4-laser LSR Fortessa (BD Biosciences, Mountain View, CA, USA). Data were analyzed using FACS DiVa software (version 8.1).
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Tracking iNKT Cell Expansion Dynamics
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