CD11c fluorescein isothiocyanate (FITC)-, CD11c allophycocyanin- (APC), Ia-Ie phycoerythrin (PE), IA-b APC, CD103 PE, TLR4 PE, F4/80 APC, CD11b PE, CD8 PE and CD4 APC were purchased from BD Biosciences and Biotin-conjugated PDL1+, PD-L2+ and APC-conjugated Streptavidin were from BioLegend. For surface staining, cells were washed with 0.5% bovine serum albumin in PBS (pH 7.4) and stained at 4°C for 15 min. in the presence of CD16/32 blocking antibodies. To characterize Th1, Th2 and Th17 cells, lung leukocytes were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA), 500 ng/ml ionomycin and 10 μg/ml brefeldin A (BFA) for 6 h in vitro and stained for surface markers as above followed by fixation and permeabilization with Cytofix/Cytoperm (BD Biosciences) for 20 mins at 4°C, washed in permeabilization buffer (BD Biosciences), and stained for 1 hour with IFNγ, IL-4 and IL-17A PE antibodies (BD Biosciences). Cells were characterized using flow cytometry (Accuri C6). Data were analyzed in FCS Express 4 Software, De Novo (CA).
Cd103 pe
Manufactured by BD
Sourced in United States
CD103-PE is a fluorescently labeled antibody that binds to the CD103 antigen. CD103 is a cell surface receptor expressed on certain immune cells. The PE (Phycoerythrin) fluorescent label allows for the detection and analysis of CD103-positive cells using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Permeabilization buffer, by BD (1 mentions)
Tlr4 pe, by BD (1 mentions)
F4 80 apc, by BD (1 mentions)
Cd11c fluorescein isothiocyanate fitc, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Cd11b pe, by BD (1 mentions)
Cd8 pe, by BD (1 mentions)
Cd4 apc, by BD (1 mentions)
Cd69 fitc, by BD (1 mentions)
Cd31 fitc, by BD (1 mentions)
Ccr7 apc, by BD (1 mentions)
Cd95 fitc, by BD (1 mentions)
Pd 1 pe, by BD (1 mentions)
Cd4 apc h7, by BD (1 mentions)
Cd127 apc, by BD (1 mentions)
Cd45ra percp cy5, by BD (1 mentions)
Cd45r0percp cy5, by BD (1 mentions)
Facsverse cytometer, by BD (1 mentions)
Cd8 pe cy7, by BD (1 mentions)
Cd38 pe, by BD (1 mentions)
8 protocols using cd103 pe
1
Multiparametric Characterization of Lung Immune Cells
2
Immunophenotyping of T-cell Subsets in HIV Infection
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Lymphocyte surface phenotypes were evaluated by flow cytometry on fresh peripheral blood of HIV-infected women alone, using fluorochrome-labeled antibodies: L/D-BV510 (Miltenyi Biotech, Germany); CD4-APC-H7, CD8-PE-Cy7, CD38-PE, CD45R0-PerCPCy5.5, CD45RA-PerCPCy5.5, CD127-APC, CD31-FITC, CCR7-APC, CD103-PE, CD95-FITC, CD69-FITC, PD1-PE (BD Biosciences, Palo Alto, CA).
T-cell subsets were defined as: naive CCR7+ CD45RA+, central memory CCR7+CD45RA−, effector memory CCR7− CD45RA−, and terminally differentiated CCR7− CD45RA+ subsets. Besides, we measured: activation (CD45R0/CD38/CD69), apoptosis (CD95), exhaustion (PD-1), IL7R (CD127), and Recent Thymic Emigrants (CD31 or CD103) on both CD4 and CD8 T-cell subsets.
Briefly, 1 × 106 peripheral blood mononuclear Cells were stained with the appropriate antibodies for 20 minutes at 4°C in the dark, washed and then acquired using FACSVerse cytometer (BD Biosciences, Palo Alto, CA).
T-cell subsets were defined as: naive CCR7+ CD45RA+, central memory CCR7+CD45RA−, effector memory CCR7− CD45RA−, and terminally differentiated CCR7− CD45RA+ subsets. Besides, we measured: activation (CD45R0/CD38/CD69), apoptosis (CD95), exhaustion (PD-1), IL7R (CD127), and Recent Thymic Emigrants (CD31 or CD103) on both CD4 and CD8 T-cell subsets.
Briefly, 1 × 106 peripheral blood mononuclear Cells were stained with the appropriate antibodies for 20 minutes at 4°C in the dark, washed and then acquired using FACSVerse cytometer (BD Biosciences, Palo Alto, CA).
3
Comprehensive Multiparameter Gut Immune Profiling
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Fluorochrome-conjugated monoclonal antibodies (mAbs) used in this study were CD45-PerCP and -Alexa Fluor 700, EpCam-FITC and -BV510, CD3-PerCP-Cy5.5, CD4-PE, -PE-Cy7, -BV786, and -AlexaFluor 700, CD8-APC-Cy7, CD45RA-BV605, HLA-DR-FITC, and -APC-Cy7, CD38-APC and -PE-Cy7, CD25-APC and -BB515, CD127-PE-CF594, CD103-PE, CXCR5-Alexa Fluor 647, CD16-BV421, and CD56-APC from BD Biosciences (San Jose, CA, USA); CD4+ 5RA-ECD (clone 2H4) from Beckman Coulter (Hialeah, FL, USA); CD127-eFluor450 and CD62L-APC-eFluor780 from eBioscience (San Diego, CA, USA); PD-1 (PE or APC, clone EH-12) and CD127-BV421 and -PE-Cy7 from BioLegend (San Diego, CA, USA); and IgA-PE and CD161-APC and -PE-Cy7 (Miltenyi Biotec, Germany). All antibodies were used according to the manufacturers’ directions.
For detection of surface markers, gut biopsy cells were stained with fluorochrome-conjugated antibodies for 15 min at room temperature, washed once with PBA [Dulbecco’s phosphate-buffered saline (DPBS) containing 0.5% BSA and 0.1% sodium azide], and resuspended in 0.5% paraformaldehyde (Electron Microscopy Sciences, PA, USA) in DPBS (PFA) for fixation, as previously described for human immunophenotyping (40 (link)). Intracellular staining for Ki-67 was performed as previously described (40 (link)), and intracellular staining for Bcl-6 was performed as previously described (31 (link)).
For detection of surface markers, gut biopsy cells were stained with fluorochrome-conjugated antibodies for 15 min at room temperature, washed once with PBA [Dulbecco’s phosphate-buffered saline (DPBS) containing 0.5% BSA and 0.1% sodium azide], and resuspended in 0.5% paraformaldehyde (Electron Microscopy Sciences, PA, USA) in DPBS (PFA) for fixation, as previously described for human immunophenotyping (40 (link)). Intracellular staining for Ki-67 was performed as previously described (40 (link)), and intracellular staining for Bcl-6 was performed as previously described (31 (link)).
4
Comprehensive Immune Cell Profiling
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Cells (1 × 106) were stained on ice for 20 min with combinations of the following antibodies: CD3 (APC-Cy7 and Qdot 605) (Life technologies, Grand Island, NY, USA); CD4 (V450 and V500) (BD Biosciences, San Jose, CA, USA); CD25 (PE-Cy7 and APC-Cy7), CD120b/tumor necrosis factor 2 (TNFR2) (PE), latency associated peptide (LAP) (Per-CP), cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) biotin or their respective immunoglobulin isotypes (all eBioscience, San Diego, CA, USA). For intracellular staining of Foxp3 (APC) and Ki67 (FITC) (eBioscience, San Diego, CA, USA), cells were first permeabilized according to the manufacturer’s instructions. The following antibodies were used to identify CD103+ DC: CD103 (PE) (BD Biosciences), CD11c (APC) and MHCII (APC-eFluor 780) (eBioscience), CD11b (AF700) and CD86 (Brilliant Violet Blue) (BioLegend), and Live/Dead cell stain kit-Aqua (Invitrogen). Acquisition was on an LSR II flow cytometer (BD Biosciences, San Jose, CA, USA) and analysis was performed using FlowJo (Tree Star, Ashland, OR, USA).
5
Investigating Murine Lymph Node Dendritic Cells
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Mouse ears were treated with Aldara cream, containing 5% imiquimod, and 3 and 21 h later mice received into the ear pinna an injection of 12.5 μg LecB in 15 μl saline water. For DEC‐205 targeting, 1 μg anti‐DEC‐205‐A647 antibody diluted in 15 μl PBS was injected into ears pinna prior to Aldara cream application (Flacher et al, 2012 (link)). Auricular LNs were harvested 72 h afterwards. For single cell preparation, LNs were cut into small pieces and digested with 1 mg/ml collagenase D (Roche) and 0.1 mg/ml DNase I (Roche) in RPMI cell culture medium containing 2% FCS for 1 h at 37°C under agitation. For flow cytometry staining the antibodies were: CD11c‐PE‐Cy7, CD103‐PE (BD Pharmingen), MHC‐II‐AF700, CD205‐AF647 (BioLegend), and CD207 (Langerin)‐AF488 (Eurobio).
6
Lung Cell Isolation and Flow Cytometry
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An amount of 2 μg of anti-mouse CD45-BV786 antibody (BD Biosciences, Franklin Lakes, NJ, USA) was diluted in sterile PBS and administered intravenously in the tail vein 10 min before anesthesia. CD 45+ is a circulating lymphocyte, and CD45− is a tissue-resident cell. In total, 40 U/mL DNase I and 1 mg/mL Collagenase IV enzyme were measured to a volume of 2 mL and incubated with lung tissue at 37 °C for 30 min. The tissue was then homogenized using a dissociator, filtered through a 70 μM cell strainer, and subjected to RBC lysis to obtain single lung cells. After preparing the lung cells to a concentration of 5 × 106 cell/mL, they were washed with PBS and live-cell staining was performed using fixable viability stain 700 (FVS700) (BD Biosciences, Franklin Lakes, NJ, USA). After treating lung cells with Fc BlockTM reagent, the surface markers CD44-BV421, CD69-BV605, CD103-PE, CD62L-APC, CD4-FITC, CD3-PE-Cy7, and CD8-BB700-per cycle 5.5 ((BD Biosciences, Franklin Lakes, NJ, USA)) were used for staining. The flow cytometry gating strategy was performed according to Supplementary Materials Figure S1 , based on a previous study [45 (link)]. FACS Aria Fusion from BD Biosciences was used, and all results were derived using FlowJo software v10.
7
Identification of Dendritic Cell Subsets
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One percent FITC in 1:1 acetone: dibutyl phthalate was painted on the dorsal side of the ears of WT and KO mice. Ear-draining auricular LNs were collected for analysis 20 and 48 h after the painting. dLNs were digested for 30 min in 100 micrograms/ml DNase I and 0.5 mg/ml Collagenase P at 37°C. EDTA was added for the final 5 min incubation. The single-cell suspensions were stained for flow cytometry: CD45-BV650, CD103-PE, CD11c-PerCP-Cy5.5 or CD11C-VB421, CD11b-eFluor450 or CD11b-APC-Cy7, and MHC II-PE-Cy7 or MHC II- PerCP-eFluor 710 (all from BD Biosciences) for 20 min, washed and fixed with 4% PFA. Thereafter, the cells were washed with 0.5% saponin buffer and stained with Langerin-Alexa Fluor 647 (Dendritics, 929F3.01) for 20 min at RT. The samples were measured with LSR Fortessa flow cytometer (BD) and analyzed with FlowJo software (Treestar). Dendritic cell populations were defined among CD11c+MHCIIhi events as follows: Langerin+CD103- (Langerhans cells), Langerin+CD103+ (CD103+ dermal dendritic cells, DDC), Langerin-CD11b+ (CD11b+ DDC) and Langerin-CD11b- (double negative DDC).
8
Immunophenotyping of Immune Cells
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The following anti-human antibodies were used: CD11c APC (Clone B-ly6), CD80 PE (Clone L307.4), CD86 PE (Clone 2331 (FUN-1)), HLADR PerCP (Clone L243 (G46-6)), CD4 PerCP (Clone SK3), CD8 PerCP, CD103 PE, CD107a PE, and Granzyme B Alexa 647 were all from BD Biosciences (San Jose, CA, USA). An isotype antibody was used as a negative control (BD Biosciences, San Jose, CA, USA). FACS analysisflow cytometry was performed using FACScalibur (Becton-Dickenson, San Jose, CA, USA), and analyzed using FlowJo software (Tree Star, Inc.).
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