Cells were dislodged from the curette by repeated pipetting with PBS+. Cells were spun down (440 x g for 5 minutes) and resuspended in PBS++ containing LIVE/DEAD® Fixable Aqua Dead Cell Stain (ThermoFisher). After 15 minute incubation on ice, an antibody cocktail containing Epcam-PE, HLADR-PECy7, CD16-APC, CD66b-FITC (all Biolegend), CD3-APCCy7, CD14-PercpCy5.5 (BD Biosciences) and CD45-PACOrange (ThermoFisher) was added to the cells. Following a further 15 minute incubation on ice, cells were filtered over a 70 μm filter (ThermoFisher). Cells were spun down (440 x g for 5 minutes), resuspended in PBS++ and acquired on a flow cytometer (LSRII, BD). Samples with less than five hundred events (15% of all measured) were excluded from further analysis. Nasal washes were similarly processed excluding the dislodging step. To 100 μL heparinized blood, the viability dye and antibodies were directly added sequentially. After the final incubation step, BD FACS lysing solution was used to remove red blood cells according to manufacturer’s instruction. Flow cytometry data was analysed using Flowjo V. 10 (Treestar).
Cd66b fitc
Manufactured by BioLegend
Sourced in United States
CD66b-FITC is a fluorescently labeled antibody that binds to the CD66b surface antigen. CD66b is a member of the carcinoembryonic antigen (CEA) family and is expressed on the surface of granulocytes, including neutrophils, eosinophils, and basophils. The FITC fluorescent label allows for the detection and identification of cells expressing CD66b using flow cytometry or other fluorescence-based methods.
Automatically generated - may contain errors
Lab products found in correlation
Epcam pe, by BioLegend (4 mentions)
Hla dr pe cy7, by BioLegend (4 mentions)
Cd45 pacorange, by Thermo Fisher Scientific (4 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (3 mentions)
Cd3 apc cy7, by BD (3 mentions)
Cd14 percp cy5, by BD (3 mentions)
70 μm filter, by Thermo Fisher Scientific (2 mentions)
Cd16 apc, by BioLegend (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Cd19 bv650, by BioLegend (2 mentions)
Facs lysing solution, by BD (1 mentions)
Live dead fixable near ir dye, by Thermo Fisher Scientific (1 mentions)
Cd16 apc, by BD (1 mentions)
Facs lysis buffer, by BD (1 mentions)
Tigit pe cy7, by BioLegend (1 mentions)
Live dead fixable violet dead cell stain, by Thermo Fisher Scientific (1 mentions)
Cd3 apch7 sk7, by BD (1 mentions)
Annexin 5 apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Annexin 5 pe, by Thermo Fisher Scientific (1 mentions)
10 protocols using cd66b fitc
1
Multiparametric Flow Cytometry Analysis
2
Multiparametric Flow Cytometry Analysis of Neutrophil Activation Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies purchased from Biolegend were used: CD66bFITC (involved in respiratory burst, adhesion molecule, present in the membrane of specific granules); CD15PE (involved in cell-cell interactions, phagocytosis, stimulation of degranulation, and respiratory burst); CD63PE-Cy7 (marker for release of azurophilic granules); CD33PE-Cy5 (marker of immature neutrophils, adhesion molecule); CD16AF700 (Marker of mature granulocytes, involved in degranulation). In addition, Zenon Pacific blue conjugated Arginase-1 (Hycult Biotechnology/Invitrogen) was used to detect intracellular arginase 1. The Live/Dead Fixable Near-IR dye (Invitrogen) was used to distinguish live and dead cells.
Cells isolated from cord blood, PlaCs and PBMC were washed and incubated with 20 µl FcR blocking reagent for 5 minutes and stained for 20 minutes with cell surface markers at room temperature. Cells were washed with FACS medium and fixed with cold 4% formaldehyde on ice for 20 minutes.
For intracellular staining, 0.5% saponin was used to permeabilize the cells for 20 minutes at room temperature and then stained with arginase 1 Pacific blue conjugated antibody for a further 20 minutes. The cells were washed and analyzed immediately with an LSRII flow cytometer (BD Bioscience). The results were analyzed using FlowJo v9.6.2 (Tree Star, Ashland, OR).
Cells isolated from cord blood, PlaCs and PBMC were washed and incubated with 20 µl FcR blocking reagent for 5 minutes and stained for 20 minutes with cell surface markers at room temperature. Cells were washed with FACS medium and fixed with cold 4% formaldehyde on ice for 20 minutes.
For intracellular staining, 0.5% saponin was used to permeabilize the cells for 20 minutes at room temperature and then stained with arginase 1 Pacific blue conjugated antibody for a further 20 minutes. The cells were washed and analyzed immediately with an LSRII flow cytometer (BD Bioscience). The results were analyzed using FlowJo v9.6.2 (Tree Star, Ashland, OR).
3
Nasal Immunophenotyping by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunophenotyping of nasal cells obtained by curettes was performed as previously described52 (link). In brief, cells were dislodged from curettes and stained with LIVE/DEAD Fixable Violet Dead Cell Stain (Thermo Fisher Scientific) and an antibody cocktail containing Epcam-PE (9C4; BioLegend), HLADR-PECy7 (L243; BioLegend), CD16-APC (3G8; BioLegend), CD66b-FITC (G10F5, BioLegend), CD3-APCH7 (SK7; BD Biosciences), CD14-PercpCy5.5 (MφP9, BD Biosciences) and CD45-PACOrange (HI30, Thermo Fisher Scientific). Whole blood was stained for 15 min at room temperature with TIGIT-PECy7 (A15153G, BioLegend) and CD16-APC, followed by 2 × 10 min incubation steps with FACSLysis buffer (BD Biosciences) to remove erythrocytes. Samples were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed using Flowjo X (Treestar). Fluorescent minus one controls for each of the included antibodies were used to validate results. For the LAIV and control cohorts, but not the additional validation cohort (Supplementary Fig. 5c ), 84 of 553 samples (15.2%) had less than 500 immune cells or 250 epithelial cells and were excluded from further analysis.
4
Multiparametric Analysis of Neutrophil Biology
Check if the same lab product or an alternative is used in the 5 most similar protocols
FACS: PMNs (5 × 105) were suspended in FACS-buffer (PBS, 10% FBS, 0.1% NaN3) and stained for 30 min at RT using CD66B-FITC (Biolegend), CD34-PE (Caltag) and CD61-AF647 (Biolegend). Acquisition: Accuri-C6-Plus (Becton Dickinson). PMN (10,000 events) were analyzed using Flow Jo (version 10).
Late apoptosis : isolated neutrophils were incubated in 100 μl annexin binding buffer containing phycoerythrin-conjugated Annexin V (Annexin V-PE) and 7-amino-actinomycin D (7-AAD) (Annexin-V Apoptosis Detection Kit, ThermoFisher Scientific) for 15 min at room temperature (RT) in the dark. After incubation, 400 μl annexin binding buffer was added, and samples were measured immediately on a BD Accuri™ C6 FACS (BD Biosciences). The Annexin V-PE+/7-AAD+ populations were taken as measurements of late apoptotic cells. Data were analyzed using FlowJo v10 software (FlowJo, LLC).
NETs quantification : PMN (2.5 × 104) were incubated with 0.2 μM SytoxGreen (Life Technologies), 37°C, 5% CO2, 3 h (13 (link)). PMA (25 nM) was used as the positive control. Results are displayed as mean fluorescence intensity (MFI) measured using a Synergy H1-Hybrid-Reader (Biotek). Excitation: 485 nm/emission: 535 nm.
+ Expand
5
Nasal B and MAIT Cell Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunophenotyping of nasal B and MAIT cells obtained by curettes was performed as described previously (29 (link)). In brief, cells were dislodged from curettes and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific) and an antibody cocktail containing, among others, Epcam-PE, HLADR-PE/Cy7, CD66b-FITC, CD19-BV650 (all BioLegend), CD3-APC/Cy7, CD14-Percp/Cy5.5 (BD Biosciences), and CD45-PACOrange (Thermo Fisher Scientific) for B cells, while the cocktail for MAIT cells also included CD8–BV785 and TCRva7.2-BV711 or TCRva7.2–PE/Texas Red and CD45-BV510 (BioLegend). Samples were acquired on a BD LSR II flow cytometer and analyzed using FlowJo X. Fluorescence-minus-one controls for each of the included antibodies were used to validate results during setup of all of the panels used. Samples with less than 500 immune cells or 250 epithelial cells (11.9% of all nasal samples) were excluded from further analysis. A full list of all antibodies used for flow cytometry is provided in Supplemental Table 2 ).
6
Immunophenotyping of Tumor-Infiltrating Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were dislodged from the curette by repeated pipetting with PBS+ as described previously (19 (link), 27 (link)). Cells were spun down at 440 × g for 5 min and resuspended in PBS++ containing LIVE/DEAD fixable aqua dead cell stain (Thermo Fisher). After 15 min incubation on ice, an antibody cocktail, which included EpCam-PE, HLADR-PECy7, CD66b-FITC, CD19-BV650 (all BioLegend), CD3-APCCy7, CD14-PercpCy5.5 (BD Biosciences), and CD45-PACOrange (Thermo Fisher), was added to the cells. Following a further 15-min incubation on ice, cells were filtered over a 70-μm filter (Thermo Fisher). Cells were spun down (440 × g for 5 min), resuspended in PBS containing 0.5% heat-inactivated fetal bovine serum and 5 mM EDTA (Invitrogen), and acquired on a flow cytometer (LSR II; BD). All cells per tube were acquired, and samples with less than 500 immune cells or 250 epithelial cells were excluded from further analysis (9/40 samples; 22.5%). The numbers of acquired cells per population were used as counts. Flow cytometry data were analyzed using FlowJo version 10 (Tree Star Inc., San Carlos, CA, USA).
7
Neutrophil Fc Receptor Blocking and PP13 Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fc receptor blocking on neutrophils was performed for 10 min (FcR binding inhibitor antibody, Invitrogen Life Technologies) in staining buffer (PBS with 5% FBS and 0.1% sodium azide) on ice. Cells were washed once and then stained with CD66b-FITC (BioLegend, clone: G10F5), CD11b-PE (BioLegend, clone: ICRF44), and PD-L1-APC (eBioscience, clone: MIH1) for 20 min on ice in dark. To measure the binding of recombinant PP13 to the surface of neutrophils, 2 × 105 cells were initially washed in PBS containing 1% BSA. Recombinant PP13, which we conjugated with CF488 fluorophore using the Mix-n-Stain CF488 kit (Sigma-Aldrich) according to the manufacturers protocol, was incubated for 60 min on ice or at 37°C. After washing, Fc receptors were blocked with human FcR blocking reagent (Miltenyi Biotec) for 5 min on ice. Anti-CD66b-APC antibody (Biolegend) was used to stain neutrophils. In both cases, stained neutrophils were washed twice and then acquired on a CytoFLEX device (Beckman Coulter) by collecting data from 50,000 cells. Data was analyzed using FlowJo v10 software.
8
Optimizing scRNA-seq Sample Preparation from Synovial Fluid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare the highest quality sample for single cell RNA sequencing (scRNA-seq), patients who had the highest percentage of viable, non-neutrophil SFCs were selected, with 3 patients having septic arthritis, and 3 having inflammatory arthritis, regardless of crystal status. As SF in non-pathologic states lacks sufficient cellularity for scRNA-seq analysis, healthy patients were not included. SFCs were thawed in a 37°C water bath and diluted in 4 mL Fluorescence Activated Cell Sorting (FACS) buffer and centrifuged at 40°C at 1400 rpm. Supernatant was gently decanted, and cells were resuspended in 50 μL of ice cold FACS buffer before staining 1:1000 with DAPI and 5 μl/reaction of: CD244/APC (Clone C1.7), CD11b/BV695 (Clone ICRF44), CD66b/FITC (Clone G10F5) and CD56/PE (Clone 5.1H11) (BioLegend). Cells were incubated in the dark at 40°C for 30 minutes, washed once in ice cold FACS buffer, and resuspended in 50 μL of FACS buffer. Samples were then sorted on a Sony MA900 (San Jose, CA) with 100 μm sorting chip, sorting out CD66b+ and DAPI positive cells, then sorting on CD11b+, CD56+, or CD244+ positive cells into tubes containing cold PBS with 1% BSA. Cells were then counted for viability using trypan blue on a hemocytometer and concentrated according to 10X Chromium 3’ kit guidelines.
9
Defining Immune Cell Populations in PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frequencies of LDG and monocytes within PBMCs as well as the cell number and purity of neutrophils were defined in single cell suspensions of 1x106 cells/mL. Surface staining for anti-human: CD15-APC (BioLegend), CD14-PE (BioLegend), CD16-APC-Cy7 (BioLegend) and CD66b-FITC (BioLegend) was performed for 15 min at 4 °C. Cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA). Viability dye (Zombie NIR, BioLegend) was used to determine dead cells.
Intracellular cytokine levels from CD4+ cells, were determined by stimulating PBMCs with 5 ng/mL phorbol 12-myristate 13-acetat (Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) for 5 h upon addition of Brefeldin A (BioLegend). Stimulation was stopped and cells were fixed with 4% PFA, treated with 0.05% saponin (Sigma-Aldrich) and stained intracellularly with anti-human: CD4-BV650 (BioLegend), IFN-y-PB (BioLegend), IL-17-PcP (BioLegend) and IL-22-PE-Cy7 (BioLegend) (all 1:100). Expression levels were determined via flow cytometry (BD FACS Canto II), obtained data analysed with FlowJo V10.1 and statistics calculated in GraphPad Prism 5.0.
Intracellular cytokine levels from CD4+ cells, were determined by stimulating PBMCs with 5 ng/mL phorbol 12-myristate 13-acetat (Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) for 5 h upon addition of Brefeldin A (BioLegend). Stimulation was stopped and cells were fixed with 4% PFA, treated with 0.05% saponin (Sigma-Aldrich) and stained intracellularly with anti-human: CD4-BV650 (BioLegend), IFN-y-PB (BioLegend), IL-17-PcP (BioLegend) and IL-22-PE-Cy7 (BioLegend) (all 1:100). Expression levels were determined via flow cytometry (BD FACS Canto II), obtained data analysed with FlowJo V10.1 and statistics calculated in GraphPad Prism 5.0.
10
sTREM-1 Quantification and Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The concentration of sTREM-1 was measured by duplicate in sera using a commercially enzyme-linked immunosorbent assay (ELISAs; Human TREM-1 Quantikine ELISA Kit) and quality control of ELISA assays was made according to the manufacturer's instructions.
The minimal level of detection of ELISA assay for sTREM-1 was 4.5 pg/ml. The peripheral blood mononuclear cells (PBMCs) were isolated by Fycoll-paque; the TREM-1 expressed in the membrane of monocytes and neutrophils was determined with the fluorescenceconjugated mouse anti-human antibodies anti-CD64-PeCy7, CD66b-FITC, CD16-PeCy5 and CD354-PE (Biolegend, San Diego, CA, USA) and the fluorescence was detected by flow cytometry using the Attune-NxT cytometer, acquired data was analyzed using the FlowJo X software (TreeStar) at the Infectious Diseases Research Laboratory, Facultad de Medicina, UNAM.
The minimal level of detection of ELISA assay for sTREM-1 was 4.5 pg/ml. The peripheral blood mononuclear cells (PBMCs) were isolated by Fycoll-paque; the TREM-1 expressed in the membrane of monocytes and neutrophils was determined with the fluorescenceconjugated mouse anti-human antibodies anti-CD64-PeCy7, CD66b-FITC, CD16-PeCy5 and CD354-PE (Biolegend, San Diego, CA, USA) and the fluorescence was detected by flow cytometry using the Attune-NxT cytometer, acquired data was analyzed using the FlowJo X software (TreeStar) at the Infectious Diseases Research Laboratory, Facultad de Medicina, UNAM.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!