Cells were stained with specific antibodies or control isotypes using conventional protocols. The following anti-mouse antibodies were used for FACS analysis of MDSCs: CD11b/PECy5, Gr1/PE, Gr1/APC, CD62L/FITC, F4/80/FITC, CD11c/PE, and MHC CI OVA peptide/Biotin from eBiosciences (San Diego, CA); as well as CD124/PE, CD40/FITC, CD86/FITC, IA-IE/FITC, Ly6C/FITC and Ly6G/PE that were purchased from BD Biosciences (San Jose, CA). Lymphocytes characterization was performed with the next specific antibodies: CD8/PE and CD8/Biotin, CD69/PECy5, CD62L/FITC, CD247/PE, CD4/PE, CD4/PECy5, CD25/PECy5, Foxp3/Biotin, IFN-γ/FITC, IL-4/PE and IL-17/PE, all from eBiosciences. FITC-conjugated Streptavidin was purchased also from eBiosciences. In all cases, cells were previously incubated with anti-mouse FcγR 2.4G2 ascites (HB-197; ATCC) to reduce the non-specific binding. For intracellular staining, cells were fixed and permeabilized, according to the manufacturer’s protocol, with either Foxp3 Staining Buffer Set or IC Fixation and 10X Permeabilization Buffers from eBiosciences. Cells were acquired using both FACScan (BD Biosciences) and Gallios (Beckman Coulter, Miami, FL) flow cytometers and analyzed with Kaluza 1.2 (Beckman Coulter) and FlowJo 5.7.2 (Tree Star Inc., USA) softwares.
Cd62l fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD62L-FITC is a fluorescently-labeled antibody that binds to the CD62L (L-selectin) cell surface antigen. CD62L is a cell adhesion molecule involved in the homing and migration of lymphocytes.
Automatically generated - may contain errors
Lab products found in correlation
Cd44 pe, by Thermo Fisher Scientific (5 mentions)
Cd69 fitc, by Thermo Fisher Scientific (4 mentions)
Cd25 pe, by Thermo Fisher Scientific (3 mentions)
Cd8 percp cy5, by Thermo Fisher Scientific (3 mentions)
Gl 7 fitc, by Thermo Fisher Scientific (3 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (3 mentions)
Cd4 apc, by Thermo Fisher Scientific (3 mentions)
Cd3 alexa780, by Thermo Fisher Scientific (2 mentions)
Cd8 v500, by Thermo Fisher Scientific (2 mentions)
Cd103 pe, by Thermo Fisher Scientific (2 mentions)
Cd197 percpcy5, by Thermo Fisher Scientific (2 mentions)
Cd4 qdot655, by Thermo Fisher Scientific (2 mentions)
Live dead fixable blue dead cell stain, by Thermo Fisher Scientific (2 mentions)
Cd45 pe cy7, by Thermo Fisher Scientific (2 mentions)
Calibur, by BD (2 mentions)
Cd8 pe, by Thermo Fisher Scientific (2 mentions)
Cd4 pe, by Thermo Fisher Scientific (2 mentions)
Gr 1 pe, by Thermo Fisher Scientific (2 mentions)
F4 80 fitc, by Thermo Fisher Scientific (2 mentions)
Cd4 pe cy5, by Thermo Fisher Scientific (2 mentions)
16 protocols using cd62l fitc
1
FACS Analysis of Myeloid-Derived Suppressor Cells
2
Phenotypic Characterization of Menstrual Blood Cells
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Phenotypic characterization of menstrual blood cells (MBC), mononuclear cells isolated from tissue, and PBMC employed cell-surface markers CD3-Alexa780 (eBioscience, San Diego, CA), CD8-V500, CD45-PEcy7, CD62L-FITC, CD69-APC, CD103-PE (αEβ7), CD197-PercpCy5.5 (CCR7), CD4-Qdot655 (ThermoFisher, Waltham, MA), and fluorescent LIVE/DEAD Fixable Blue Dead Cell Stain (Molecular probes, Invitrogen, CA). Stained cells were acquired using a BD Calibur flow cytometer (Becton Dickinson [BD], San Jose, CA) and analyzed with FlowJo Version 9.9 software for Mac (TreeStar, San Carlos, CA). We ran at least 100,000 gated lymphocytes for each stained specimen. Fluorescence minus one (FMO) on cells stimulated with staphylococcus enterotoxin B (SEB) was used to set gates for the TrM cells (CD62L-, CCR7-, CD103+, CD69+). All antibodies were obtained from BD Biosciences, San Jose, California unless otherwise noted.
3
Menstrual Blood Cells Phenotypic Characterization
4
Multiparameter Flow Cytometry Analysis
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Splenocytes, mesenteric lymph node cells, and inguinal lymph node cells were isolated from the indicated mouse and then stained with anti-mouse CD4-PE/Cy7, CD8-Percp/Cy5.5, CD25-PE, CD69-FITC, CD44-APC-Cy7, CD62L-FITC, B220-Percp/Cy5.5, GL-7-FITC, and CD95-PE antibodies (eBioscience, San Diego, CA, USA) for 15 min at 4 °C. For TFH cell analysis, cells were stained with anti-mouse CXCR5-biotin for 30 min at 4 °C followed by anti-mouse CD44-APC/Cy7 and CD4-PE/Cy7 and streptavidin-APC staining. After fixation and permeabilization using a Foxp3 Staining Kit (eBioscience), intracellular Bcl-6 was stained using anti-mouse Bcl-6-PE (BD Bioscience, San Jose, CA, USA) for 40 min at room temperature. Cells were examined using a FACSCanto II system (BD Bioscience), and data were analyzed using FlowJo software (Treestar, Ashland, OR, USA).
5
Phenotypic Analysis of PBMCs and HDNs
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LDNs were analyzed in the fraction of PBMC. PBMCs or HDNs (5 × 105 cells) were stained in the presence of Human FcR Blocking for 30 min at 4°C and protected from light with or without the following mixture of fluorescent-labeled, anti-human antibodies: CD14-FITC or PercP-Cy5.5; CD16-PE-Cy7; CD62L-FITC (eBioscience, USA); CD15-PE-Cy5 or Pacific Blue; and CD11b-PE (Biolegend, USA). Cells were washed with PBS/BSA 1% and fixed in 4% paraformaldehyde. The samples were stored protected from light in the refrigerator until the moment of acquisition. The BD FACSAria IIu cytometer (BD Biosciences, USA) was used to acquire the samples.
6
Multiparametric Flow Cytometry of Murine Splenocytes
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A total of 1 × 106 lymphocytes were acquired and collected from mice spleen, and then 0.5 μg anti-mouse CD16/32 monoclonal antibody (Biolegend, USA) and 5 μL rat serum were used to block the Fc receptor for 10 min. For measuring the number of total CD4+ T cell and naïve CD4+ T cell, splenocytes were incubated with 1 μg CD3-APC-Cy7 (Biolegend), CD4-APC (eBioscience, Thermo Fisher Scientific, USA), TCRβ-PE and CD62L-FITC (eBioscience, Thermo) for 30 min. For detecting cell activation, cells were stained with 1 μg CD25-PE and CD69-FITC (eBioscience, Thermo). For evaluating cell surface protein expression, EGFR (Thermo)-PE (eBioscience) and Glut1 (Proteintech, China) -FITC (eBioscience) were incubated for 30 min. To measure cell death, splenic cells were stained with PI and Annexin V (BD Biosciences, San Jose, CA, USA) for 15 min at room temperature. Cells were analyzed by FACSCanto II cytometer (BD Biosciences, San Jose, CA, USA). All data were analyzed by Flowjo10 software (Tree Star, Ashland, OR, USA).
7
Multi-Dimensional Flow Cytometry Profiling
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Splenocytes, mesenteric, and inguinal lymph node cells were isolated and then stained with anti-mouse CD4-APC, CD8-PerCP-Cy5.5, CD44-PE, CD62L-FITC, GL-7-FITC, CD95-PE, and B220-PerCP-Cy5.5 antibodies (eBioscience, San Diego, CA) for 15 min at 4 °C. For TFH differentiation analysis, the cells were stained with anti-mouse CXCR5-biotin for 30 min at 4 °C followed by anti-mouse CD44-FITC, CD4-PerCP-Cy5.5, and streptavidin-APC staining. After fixation and permeabilization using the Foxp3 Staining Kit (eBioscience), anti-mouse Bcl-6-PE was stained for 1 h at room temperature. Cells were examined using the FACSCanto II system (BD Bioscience, San Jose, CA, USA) and data were analyzed using Flow Jo software (Treestar, Ashland, OR, USA). In all cases, doublets (FSC-area versus FSC-height gating) were excluded.
8
Flow Cytometry Analysis of T Cell Subsets
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Following the removal of the olfactory bulb at the indicated time points, tissue was placed in a 2 ml Wheatley Dounce Homogenizer (Fisher Scientific) with 2 ml DMEM media supplemented with high glucose, L-glutamine, and pyruvate (Life Technologies) and 10% FBS. Single cell suspensions were created and processed as previously described [21 (link)]. Briefly, 1/10 the sample homogenate was filtered using a 40-μm nylon mesh filter (Fisher), was pre-incubated with 0.8 μg Fc block (CD16/32) (eBioscience) and then was immunolabeled in 1% FBS/BSA. Total T cells were stained for CD45 eFlour 450 (clone 30-F11), CD3e FITC (clone 145-2C11), CD8a PE (clone 53-6.7), and CD4 APC (clone GK1.5) (all eBioscience). Effector (T-EM) and central memory (T-CM) cells were identified by CD45 eFlour 450, CD3e PE-Cy7, CD4 APC-Cy7, CD8a PE, CD44 APC, and CD62L FITC all from eBioscience as described [21 (link)]. HSV-1-specific T cells were identified by CD3 eFluor 450, CD8a FITC, and either gB-PE, ICP8-A488, or RRI-A488 tetramers (provided by the NIH tetramer facility) as previously described [21 (link)]. All samples were analyzed with the MacsQuant flow cytometer and MacsQuantify software (Miltenyi Biotec).
9
Multiparameter Flow Cytometry Analysis
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Splenocytes and mesenteric lymph node cells were isolated and then stained with anti-mouse CD4-APC, CD8-PerCP-Cy5.5, CD19-PE, CD44-PE, CD62L-FITC, CD69-FITC, IL-7Rα-PE, CD138-APC, PSGL-1-APC, CXCR5-PE, PD-1-FITC, GL-7-FITC, CD95-PE-Cy7, B220-Alexa647 and NK1.1-FITC (eBioscience, San Diego, CA) antibodies for 15 min at 4°C. To assess intracellular cytokine levels, cells were differentiated for 5 days and then re-stimulated with Cell Stimulation Cocktail plus Protein Transport Inhibitors reagent (eBioscience) for 5 h. Then, the cells were fixed, permeabilized, and stained with anti-mouse IFN-γ-FITC, IL-4-PE, IL-17-APC, and IL-9-APC antibodies. Intracellular Foxp3 staining was performed using the Foxp3 Staining Kit (eBioscience). To determine the purity of isolated CD4+CD25− T cells, these cells were stained with anti-CD4 and anti-CD25, and followed by FACS analysis.
10
Isolation and Analysis of Murine Immune Cells
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DMBA and BP were purchased from AccuStandard, Inc (New Haven, CT). RPMI 1640 was purchased from Sigma Chemical (St Louis, MO). Collagenase (Type 1) was purchased from the Worthington Biochemical Corp. (Lakewood, NJ). The CFU‐preB (M3630) media were obtained from StemCell Technologies (Vancouver, BC, Canada). Fetal bovine serum was obtained from Atlanta Biologicals (Flowery Branch, GA). The following monoclonal antibodies (mAbs) for FACS analysis of splenocyte and thymocyte cell populations were purchased from BD Pharmingen (San Jose, CA): CD45/B220‐Percp–, Gr‐1‐fluorescein isothiocyanate (FITC), CD4‐APC, CD8‐Percp, CD8‐PeCy7, CD62L‐FITC, and CD44‐PE, whereas F4/80 (FITC), Sca‐1(PE) and c‐kit (FITC) were obtained from eBioscience (San Diego, CA).
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