Cells were re-suspended in PBS containing 10% fetal bovine serum (FBS) and stained with following antibodies to analyze DC subsets in PBMC: CD3-PE-Cy™7, CD14-Alexa Fluor® 700, CD19-PE-Cy™7, CD56-PE-Cy™7, CD123-PerCP-Cy5.5, CD11c-APC and HLA-DR- APC-Cy7 (BD Bioscience). For detection of intracellular cytokine (ICC), Brefeldin A was added into culture in the last 6 hours of incubation. Cells were surface stained with subset markers and made permeable with Cytofix/cytoperm solution (BD Bioscience). Cells were then stained with following antibodies: IL-6-PE, TNF-α-Pacific Blue™, IL-1β-FITC, IL-10-PE (Biolegend) and IL-12p40/70-PE (Miltenyi Biotech). Samples were analyzed using LSRII Flow cytometer (BD Biosciences) and the data were analyzed using FlowJo software (Tree Star, Inc.)
Cd14 alexa fluor 700
Manufactured by BD
Sourced in Canada
CD14-Alexa Fluor 700 is a fluorescently-labeled antibody that targets the CD14 cell surface antigen. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd56 apc, by BD (4 mentions)
Cd3 fitc, by BD (3 mentions)
Cd19 v500, by BD (3 mentions)
Cd19 pe cy7, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Cd3 pe cy7, by BD (2 mentions)
Cd16 bv711, by BD (2 mentions)
Cd19 alexa fluor 700, by BD (2 mentions)
Cd56 pe cy7, by BD (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cd25 bv605, by BD (2 mentions)
Pe cy7 cd196, by BD (2 mentions)
Bv421 cd194, by BD (2 mentions)
Pe cf594 cd183, by BD (2 mentions)
Cd127 pe, by BD (2 mentions)
Cd4 apc cy7, by BD (2 mentions)
Percp cy5.5 cd8, by BD (2 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Hla dr apc cy7, by BD (1 mentions)
12 protocols using cd14 alexa fluor 700
1
Profiling Dendritic Cell Subsets in PBMC
2
Phenotyping of Cytokine-Producing Monocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were cultured in RPMI-1640 media (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), with or without LPS (100 ng/mL, STEMCELL Technologies, Vancouver, Canada) and Golgiplug (BD Biosciences, San Diego, CA, USA) for 3 h. The cells were surface-stained with CD45-BV786, CD14-Alexa Fluor 700, CD16-BV711, HLA-DR-APC-H7, TIM-3-BB515 (BD Biosciences, San Diego, CA, USA), and TIGIT-PE-Cy7 (eBioscience, San Diego, CA, USA), and intracellularly stained with antibodies against IL-10-APC, IL-1β-Pacific blue, TNF-α-BV650 (BD Biosciences), IL-6-PE (eBioscience), GM-CSF-PE-CF594 (BioLegend), and the corresponding isotype controls. Data acquisition was performed on an LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA), and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
3
Annexin V Apoptosis Detection in Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptosis was detected using Annexin V apoptosis detection kit FITC (e-Bioscience; catalog number 88-8005-72) per manufacturer’s instructions. Briefly, after harvesting THP-1 and PBMC at the indicated times, cells were centrifuged and washed once with cold PBS followed by another wash with 1X cold Annexin V Binding Buffer. To the cells (106 cells/100μl), 5μL of fluorochrome-conjugated Annexin V was added and incubated in the dark for 15 min. Cells were washed once with 1X Annexin V Binding Buffer and re-suspended in 300μL of 1X Annexin V Binding Buffer containing 5μL Propidium iodide (eBioscience). Percent apoptosis in PBMC subsets (T cells, B cells, dendritic cells, and monocytes) was determined by staining with antibodies (BD Bioscience) specific for immune cell surface markers: HLA-DR-allophycocyanin-Cy7 (Catalog number 335796), CDllc-APC (Catalog number 340544), CD19-PerCP (Catalog number 340421), CD14-Alexa Fluor 700 (Catalog number 557923), CD3-PE-Cy7 (Catalog number 563423). Samples were analyzed using an LSRII flow cytometer (BD Bioscience).
4
Characterizing Immune Cell Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh PBMCs were cultured either alone, with Pam3CysSK4 or XS15, or a mix of phytohaemagglutinin-L (PHA) and Pokeweed mitogen (PWM). Adherent and non-adherent cells were stained with mAbs: CD3-BV711, CD56-BV421, CD19-BV785, Zombie aqua (all Biolegend), CD14-Alexa Fluor 700, HLA-DR-PerCP, and CD69-APC-Cy7 (all BD). Cells were measured by flow cytometry as described above.
5
Multiparametric Flow Cytometry of Blister Leukocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blister and circulating leukocytes were enumerated and then analysed for surface marker expression on a flow cytometer (LSR Fortessa, BD Biosciences). Due to a lack of published data on blister leukocyte differentiation using flow cytometry, a novel staining and subsequent gating strategy was designed to identify individual cell populations. Leukocytes were incubated with combinations of antibodies to CD3 (APC, Clone: UCHT1, BD), CD19 (PE-Cy 7, Clone: SJ25C1, BD), CD56 (PerCP-Cy5.5, Clone: B159, BD), HLA-DR (V450, Clone: L243, BD), CD14 (Alexa Fluor 700, Clone: M5E2, BD), CD16 (FITC, Clone: 3G8, BD), CD141 (PE, Clone: M80, Biolegend), CD163 (PE, Clone: M130, BD), CD11c (PE-Cy7, Clone: B-ly6, BD), Siglec-8 (PE, Clone:7C9, Biolegend) and Annexin-V/7AAD Apoptosis Detection Kit (BD) using respective isotype antibodies and fluorescence-minus-one (FMO) controls, and compensated for dual labelling. Separation of cell subtypes was performed using a cell sorter (FACS Aria, BD Biosciences) with subtypes undergoing histological staining using a modified Wright's method (Shandon Kwif-Diff Stain Kit, Thermo Scientific). Flow cytometry analysis was completed using FlowJo software (Tree Star Inc).
6
Multiparametric Flow Cytometry for Immune Monitoring
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immune-monitoring studies, patient PBMC samples were stained with cocktail fluorescent-conjugated antibody mix diluted in PBS containing 2% FBS + 0.01% azide for 30 min at 4 °C in the dark. After a 2-step washing with PBS, cells were fixed with 2% PFA for 20 min at 4 °C. Samples were analyzed on a BD Fortessa Flow Cytometer (UV-Violet-Blue-Yellow/Green-Red 5-Laser configuration). For the first cohort (aviremic patients) the following antibodies were used: CCR5-FITC, CD158a-PE, CD57-PE-CF594, CD14-Alexa Fluor 700, CD19-Alexa Fluor 700, CXCR4-BV421, CD62L-BV605, NKG2D-BV650, CD16-BV711, CD3-BV786, CD56-BUV395 and CD4-BUV737 (BD Biosciences), CD158b-PE, CD158e/k-PE, NKp46-PE-Vio770, NKG2C-APC, CD45RA-APC-Vio770, and CD45RO-VioGreen (Miltenyi). For the second cohort (viremic patients) the similar panel of antibodies were re-used, except for additional antibodies for CXCR4-BV421 and CD107a-PE-CF594 (BD Biosciences). Cell death and viability were determined by DAPI (BD Biosciences). Data were analyzed using FlowJo software (v10.8) (BD Biosciences) with appropriate plugins for high-dimensional analysis and visualization.
7
Antigen-specific T cell responses post-PCV13 vaccination
8
Multiparametric Analysis of PBMCs
9
Comprehensive Immunophenotyping of Whole Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole blood (150μl) was immunolabeled for 15 minutes at room temperature using the following antibodies [with catalog numbers; concentration]: 5μl for each of the following APC-Cy7-CD4 [557871; 0.2mg/ml], PerCP-Cy5.5-CD8 [560662; 0.05mg/ml], Alexa Fluor 700-CD14 [557923; 0.2mg/ml], BV605-CD25 [562660; 0.025mg/ml], PE-Cy7-CD196 [560620; 0.2mg/ml], BV421-CD194 [562579; 0.2mg/ml], PE-CF594-CD183 [562451; 0.2mg/ml], PE-CD127 [557938; 0.2mg/ml], V500-CD19 [561121; 0.2mg/ml] and 20μl of the following: FITC-CD3 [555339; 0.05mg/ml], and APC-CD56 [555518; 0.025mg/ml] (all from BD Biosciences) and 35μl buffer solution (BD Canada). After the immunolabelling was completed, red blood cells were lysed (0.06M NH4Cl, 4mM KHCO3, 0.052mM EDTA), and peripheral blood mononuclear cells were washed with PBS, PFA-fixed, and analyzed using a flow cytometer (LSR Fortessa, BD Biosciences). A minimum of 300,000 events were acquired per sample. Data analysis was performed using FlowJo (Tree Star Inc).
10
Multiparameter Characterization of NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs or tissue-infiltrating lymphocytes were stained as follows. Frozen cells were thawed and resuspended in complete RPMI1640 medium (Corning, 10–040-CVR) containing 10% fetal bovine serum (Gibco, 12662029), 1% glutamine (Immundiagnostic, K7732) and 1% penicillin and streptomycin (Gbico, 15140–122). 1×106 cells were used for each panel and stained in dark at room temperature (20–25 °C) for 30 min. The following monoclonal antibodies (Abs) were used for NK cell phenotypic characterization: Pacific Blue-Vivid (Invitrogen, CA), PE-CF594-CD3 (BD Biosciences, 562280), Alexa Fluor® 700-CD14 (BD Biosciences, 557923), PE-Cy7-CD56 (BD Biosciences, 335791), APC-NKG2D (BD Biosciences, 558071), PE-NKG2C (R&D, FAB138C), PE-NKG2A (R&D, FAB1059C), APC-CD69 (BD Biosciences, 340560), PerCP-Cy5-HLA-DR (BD Biosciences, 347364) and FITC-CD38 (BD Biosciences, 555459). After staining, the PBMCs were washed twice with phosphate buffer saline and detected using a BD LSR II Fortessa flow cytometer (BD Biosciences, NJ). The data were analyzed using the FlowJo software (TreeStar, San Carlos, CA).
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!