We developed a sensitive SARS-CoV-2 neutralization assay15 (link) by incorporating WT SARS-CoV-2 spike protein or with N501Y mutation, representing the current prevalent B.1.1.7 (alpha variant) mutation in the United States, onto lentiviruses and measuring pseudoviral entry into angiotensin converting enzyme 2 (ACE2) overexpressing 293 cells (293-ACE2). To determine the half-maximal inhibitory concentration (NT50), 3-fold serially diluted serum samples from different patients were incubated with red fluorescent protein-encoding WT SARS-CoV-2 or alpha variant pseudotyped virus at 0.2 multiplicity of infection (MOI) for 1 hour at 37° C. The mixture was subsequently incubated with 293-ACE2 cells for 72 hours, after which cells were collected, washed with FACS buffer (1 × phosphate buffered saline + 2% fetal bovine serum) and analyzed by flow cytometry using BD FACSymphony A5 analyzer. Percent infection obtained was normalized for samples derived from cells infected with WT SARS-CoV-2 or alpha variant pseudotyped virus in the absence of serum. The NT50 was determined using 4-parameter nonlinear regression (GraphPad Prism 8.0).
Facsymphony a5 analyzer
Manufactured by BD
The BD FACSymphony A5 analyzer is a flow cytometry instrument designed for high-parameter analysis of single cells. It features a compact design and supports up to 20 simultaneous detection channels.
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3 protocols using facsymphony a5 analyzer
1
SARS-CoV-2 Neutralization Assay Development
2
Rhesus Macaque Whole Blood Phenotyping
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A 22-color panel was designed to examine the changes in cell phenotypes in whole blood collected from animals during the course of the study (surface staining for CD3, CD4, CD8 (two clones), CD11b, CD11c, CD14, CD16, CD20, CD25, CD28, CD45, CD49d, CD56, CD66abce, CD95, CD123, CD127, CD159 (NKG2a), CD194 (CCR4), and HLA-DR). All antibodies were selected based on cross-reactivity with rhesus macaques and fluorochrome availability. Antibody information, including clones and fluorochromes, is listed in Table S2 .
Briefly, 100 μL of fresh EDTA whole blood was stained with Live/Dead Fixable Blue dye (ThermoFisher Scientific) and surface-stained at room temperature for 30 min. BD FACS Lysing solution (BD Biosciences, San Jose, CA, USA) was then used to lyse red blood cells according to the manufacturer’s instructions. Samples were washed twice with D-PBS and resuspended in 1% ultrapure formaldehyde (Tousimis, Rockville, MD, USA). Samples were acquired on a BD FACSymphony A5 analyzer using FACSDiva 8 software. Data were analyzed by using the FlowJo (version 10.6.) gating strategy as outlined inFigure S1A,B , established by using a combination of isotype and fluorescence-minus-one (FMO) controls.
Briefly, 100 μL of fresh EDTA whole blood was stained with Live/Dead Fixable Blue dye (ThermoFisher Scientific) and surface-stained at room temperature for 30 min. BD FACS Lysing solution (BD Biosciences, San Jose, CA, USA) was then used to lyse red blood cells according to the manufacturer’s instructions. Samples were washed twice with D-PBS and resuspended in 1% ultrapure formaldehyde (Tousimis, Rockville, MD, USA). Samples were acquired on a BD FACSymphony A5 analyzer using FACSDiva 8 software. Data were analyzed by using the FlowJo (version 10.6.) gating strategy as outlined in
3
Multicolor Flow Cytometry Analysis of Macaque PBMCs
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Two staining panels were developed for analysis of macaque PBMC from these experiments. A 22-color panel was designed to examine T-cell and NK cell responses to treatment (CD2, CD3, CD4, CD7, CD8 [2 clones], CD14, CD16, CD20, CD56, NKp44, NKG2A, CD45, CD25, CD62L, CD28, CD95, CCR4, CD107a, CD127, and HLA-DR). A 16-color panel was designed to measure monocyte responses to treatment (CD3, CD4, CD11b, CD14, CD15, CD16, CD20, CD33, CD45, CD68, CD86, CCR2, CXCR4, PDL1, and HLA-DR). All antibodies were selected based on cross-reactivity with rhesus macaques and fluorochrome availability. Antibody information for both panels, including clones and fluorochromes, is listed in S3 Table
Briefly, frozen PBMCs were thawed, counted, and resuspended at 1x107 cells/ml in D-PBS without Ca2+ or Mg2+ (Thermo Fisher Scientific). Cells were aliquoted into three 12 x 75 mm polystyrene tubes, 100 μl per tube, (Unstained, 22-color panel, 16-color panel) and stained for 30 min on ice. Samples were washed with D-PBS and resuspended in 1% ultrapure formaldehyde (Tousimis, Rockville, MD). Samples were acquired immediately on a BD FACSymphony A5 analyzer using FACSDiva 8 software. Data were analyzed using FlowJo Version 10.6.
Briefly, frozen PBMCs were thawed, counted, and resuspended at 1x107 cells/ml in D-PBS without Ca2+ or Mg2+ (Thermo Fisher Scientific). Cells were aliquoted into three 12 x 75 mm polystyrene tubes, 100 μl per tube, (Unstained, 22-color panel, 16-color panel) and stained for 30 min on ice. Samples were washed with D-PBS and resuspended in 1% ultrapure formaldehyde (Tousimis, Rockville, MD). Samples were acquired immediately on a BD FACSymphony A5 analyzer using FACSDiva 8 software. Data were analyzed using FlowJo Version 10.6.
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