2x106 lung or spleen cells were resuspended in PBS and stained with Fixable Viability Dye eFluor 450 (eBiosciences) for 20 minutes, then washed and resuspended in PBS containing 1% heat-inactivated FBS, 1 mM EDTA, 10 μg/mL CD16/32 and 0.1% sodium azide. Cells were stained for 30 minutes with appropriate antibodies, fixed in BD Stabilizing Fix, and stored at 4°C until analysis on an LSR II (Becton Dickenson). Antibodies used to identify monocytes, macrophages, neutrophils, and DCs were as follows: neutrophils (CD11c-CD11b+SiglecFloLy6G+), monocytes (CD11c-CD11b+MHCII-CD64+), CD103+ cDCs (MHCII+CD11c+CD11b-CD24+CD103+), CD11b+ cDCs (MHCII+CD11c+CD11b+CD103-), and pDCs (CD11c+/-CD11b-CD103-B220+). APC-CD45, PE-Cy7-B220, and APC.780-MHCII (eBioscience), Alexa700-CD11c and PE-Cy7-SiglecF (Miltenyi Biotec), BV510-CD103, BV510-Ly6G, BV605-CD11b and PE-CD24 (Becton Dickenson), and BV711-CD64 and BV421-CD19 (BioLegend). The antibodies used to identify T cells and NK cells were as follows: APC-CD4, PerCp-Cy5.5-CD8, PE-Cy7-CD69, and PE-CD69 (Becton Dickenson) and PerCp-Cy5.5-NK1.1, FITC-CD3ε, and APC-gamma delta T cell receptor (γδ TCR) (eBiosciences). Flow cytometry data was analyzed using FlowJo version 7.6.5.
Percp cy5.5 cd8
Manufactured by BD
Sourced in United States, Canada
PerCP-Cy5.5-CD8 is a fluorochrome-conjugated antibody used for the detection and analysis of CD8-positive cells in flow cytometry applications. It is composed of the PerCP-Cy5.5 fluorescent dye conjugated to an antibody specific for the CD8 surface marker.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 fitc, by BD (4 mentions)
Cd4 apc cy7, by BD (3 mentions)
Cd3 apc, 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)
Cd19 v500, by BD (2 mentions)
Cd56 apc, by BD (2 mentions)
Cd14 alexa fluor 700, by BD (2 mentions)
Cd8 apc h7, by BD (2 mentions)
Percp cy5.5 cd4, by BD (2 mentions)
Pe cy7 cd69 or pe cd69, by BD (2 mentions)
Cytofix cytoperm plus kit, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Cd45 apc, by Thermo Fisher Scientific (1 mentions)
Pe cy7 b220, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 450, by Thermo Fisher Scientific (1 mentions)
11 protocols using percp cy5.5 cd8
1
Multiparametric Flow Cytometry of Lung and Spleen Immune Cells
2
Antigen-Specific T Cell Characterization
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The presence of antigen-specific T cells was determined by staining with PE-conjugated HLA201/peptide multimers and intracellular staining for IFNg and TNFa. Expanded T cells were activated by restimulating with T2 cells pulsed with 10 mg/mL mutant or wild-type peptide at a ratio of 1:4 T-to-T2 cells for 4 hours in the presence of Golgi Block (eBioscience). Cells were stained with custom PE-labeled MHC multimers (Immudex), Live/Dead Blue (Thermo Fisher Scientific), and PerCP-Cy5.5 CD8 (Becton Dickinson) according to the Immudex multimer staining protocol. Intracellular staining for IFNg and TNFa was performed with the Intracellular Fixation & Permeabilization Buffer Set (eBioscience).
3
Cytokine and Immune Cell Profiling
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Blood was centrifuged and serum was separated and preserved at –70 °C in a refrigerator for cytokine analysis. After hemolysis of red blood cell (RBC), the precipitate was suspended in flow cytometry buffer solution. The harvested lymph node was mechanically dissociated, and a single cell suspension was made with 40 µm nylon cell strainer. Anti-human fluorescein isothiocyanate (FITC) CD4, phycoerythrin (PE) CD40L, PerCP-Cy5.5 CD8, allophycocyanin (APC) CD40, FITC CD33, and PerCP-Cy5.5 CD19 (BD Bioscience, CA, USA) were used for staining.
4
Toll-Like Receptor Modulation Protocol
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Nuclease-resistant phosphorothioate-modified ODNs were synthesized by Suzhou Ribo Life Science Co., Ltd. (Suzhou, China). The Poly (I: C) (a ligand of TLR3), lipopolysaccharide (LPS, Escherichia coli 055: B5, a ligand of TLR4), imiquimod (IMQ, a ligand of TLR7), and CpG 685 (CpG, a ligand of TLR9) were obtained from InvivoGen (San Diego, CA, United States). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, United States). Penicillin and streptomycin, carboxyfluorescein succinimidyl amino ester (CFSE), and RPMI-1640 medium were purchased from Invitrogen (Carlsbad, CA, United States). APC-CD3, V450-CD4, Percpcy-5.5-CD8, APC-H7-CD19, anti-rabbit IgG, and appropriate isotype-controls, the Cytometric beads array kit (human IL-6/-10 Flex set), and Annexin V/PI apoptosis kit were obtained from BD Biosciences (Franklin Lakes, NJ, United States). The WST-1 Cell Proliferation Assay Kit was provided by the Beyotime Institute of Biotechnology (Jiangsu, China). Antibodies against P-P65 (serine 536)/P65, P-IκBα/IκBα, P-JAK2 (tyrosine 1008)/JAK2, P-STAT3 (tyrosine 705)/STAT3, and β-actin were provided by Cell Signaling Technology (Danvers, MA, United States) or Santa Cruz Biotechnology (Dallas TX, United States). The catalog numbers and dilution ratios of all antibodies are listed in Table 1 .
5
Multiparameter Flow Cytometry Analysis
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Before flow cytometry analysis, macrophages and T cells were incubated with 7-Amino Actinomycin D (7-AAD, eBioscience, San Diego, CA) or Fixable Viability Dye eFluor™ 780 (FVD, eBioscience, San Diego, CA) to distinguish live cells from dead cells. Only live cells were gated for further evaluation. Macrophages were stained with PE-F4/80, and T cells were stained with APC-CD3, Qdot 605-CD4 or APC Cy7-CD4, PerCP-Cy5.5-CD8, FITC-CD25, and their respective isotypes (all antibodies were purchased from BD Bioscience, San Diego, CA) at 4 °C for 30 min. To assess the cytokine levels of T cells, PE-TNF-α and APC–IFN–γ were also used for T cell staining. Stained samples were fixed with 2 % paraformaldehyde and analyzed on an LSRFortessaTM X20 flow cytometer (BD Biosciences, San Diego, CA).
6
Comprehensive Immunophenotyping of Whole Blood
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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).
7
PBMC Immune Response Assay
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Freshly isolated PBMC from CCLm group were stimulated with PMA (50 ng/ml)/ionomicyn (10−6M) for 6 h (positive control) or SLA (10 μg/ml) or rLmlRAB or rLmlRABC (10 μg/ml) for 120 h, or kept with medium alone. Cells were treated with Golgistop (BD Biosciences) for the last 6 h of culture, then washed and incubated with antibodies: FITC CD3, PerCPcy5.5 CD4, APC-H7 CD8 or PerCPcy5.5 CD8, and PE-Cy7 CD69 or PE CD69 (BD Biosciences), for 20 min at 4 °C. For intracellular IFN-γ detection, cells were fixed and permeabilized using BD Cytoperm/cytofix plus kit (BD Biosciences) according to manufacturer’s instructions and labelled with PE-anti-IFN-γ mAb (intracellular formulation) (BD Biosciences). BD™ CompBeads Set Anti-Mouse Ig, κ (Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set) (BD Biosciences) was used for compensation controls. Analysis was performed with DIVA software.
8
Influenza NP-specific CD8+ T cell analysis
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MHC‐I tetramer targeting the immunodominant epitope of the influenza nucleoprotein (DbNP366–374 – ASNENMETM, DbPA224–233 – SSLENFRAYV) was produced in‐house and conjugated to streptavidin‐APC/PE (Life Technologies Australia Pty Ltd) at a 1:250 dilution at room temperature for 1h. Cells were stained with combinations of fluorochrome‐conjugated antibodies: PerCP‐Cy5.5‐CD3 (#551163), PE‐CD8 (#561095), BV421‐I‐Ab (#562928), APC‐CD44 (#553133), FITC‐CD44 (#553133), BV711‐CD38 (#740697), PerCP‐Cy5.5‐CD8 (#551162), APC/eF780‐CD62L (#47‐0621‐82), APC‐CD11c (#17011481), PE‐Cy7‐TCR‐va2 (#560624) from BD Biosciences, USA; and PE‐Cy7‐CD38 (#102718), BV785‐PD‐1 (#329908), APC‐Cy7‐CD45.1 (#110716), FITC‐I‐Ab (#116406), Pacific Blue‐I‐Ab (#116422), FITC‐CD19 (#115506) from Biolegend, USA. AF700‐CD3 (#56003382) was purchased from Invitrogen, USA.
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).
Live/Dead‐aqua 525 was purchased from Invitrogen. Briefly, cell suspensions were stained with Live/Dead Aqua viability dye at room temperature for 10 min followed by staining with tetramer for 15 min and cell surface marker antibodies for 30 min. Cells were fixed with 1% paraformaldehyde before analysis by flow cytometry. All antibody and tetramer staining was performed at 4°C and in the dark. Samples were subsequently acquired on a Becton Dickinson LSR Fortessa or Aria III flow cytometer and data analysed by FlowJo Software (Tree Star Inc., USA).
9
Comprehensive Immune Phenotyping of PBMCs
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PBMCs were initially stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFischer Scientific, Waltham, MA, USA) for 20 min before surface staining with conjugated antibodies in washing buffer for 20 min and fixed with CellFix solution (BD Biosciences, San Jose, CA, USA). Commercial conjugated antibodies used included: BV785-CD3, BV510-CD14, BV510-CD16, BV510-CD19, PE/Dazzle594-CD39, BV711-CD103, BV421-CCR7, PE/Cy7-CD27 (BioLegend Inc, San Diego, CA, USA); PE-CD4, PerCP/Cy5.5-CD8, BV650-PD1, BB515-Tim3, APC/H7-CD45RA (BD Bioscience, San Jose, CA, USA). All samples were acquired by a four-laser BD LSRFortessa flow cytometer (BD Company, Franklin Lakes, NJ, USA) and analyzed by FlowJo software v.10.6 (FlowJo Co, San Diego, CA, USA) [Figure 1 ].
10
Flow Cytometric Analysis of T-Cell Activation
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Freshly isolated PBMC were stimulated with PMA (50 ng/ml)/ionomicyn (10−6 M) or PHA at 10 µg/ml for 6 h (positive control) or with TSLA at 10 µg/ml or with LaPSA-38S at 10 µg/ml for 120 h and, or kept with medium alone. Cells were treated with Golgistop (BD Biosciences) for the last 6 hours of culture, then washed and incubated with antibodies: FITC CD3, PerCPcy5.5 CD4, APC-H7 CD8 or PerCPcy5.5 CD8, and PE-Cy7 CD69 or PE CD69 (BD Biosciences), for 20 minutes at 4°C. For intracellular IFN-γ detection, cells were fixed and permeabilized using BD Cytoperm/cytofix plus kit (BD Biosciences) according to manufacturer's instructions and labeled with PE-anti-IFN-γ mAb (intracellular formulation) (BD Biosciences). Analysis was performed with FACSCalibur using CellQuest Pro software or FACS canto II flow cytometer using DIVA software. FITC mouse IgG1, PerCPCy5.5 mouse IgG1, mouse IgG1PE, PE mouse IgG2a (intracellular formulation) (Biosciences) were used as isotype controls for acquisition by FACSCalibur. For acquisition by FACSCanto, BD CompBeads Set Anti-Mouse Ig, κ (Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set) (BD Biosciences) were used.
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