Single-cell suspensions isolated from indicated mouse organs were stained on ice with antibodies specific for CD4 (RM4–5; eBioscience), CD5 (53–7.3; eBioscience), CD8α (53–6.7; eBioscience), CD8β (YTS 156.7.7; Absolute Antibody), CD25 (3C7; BioLegend), CD44 (IM7; eBioscience), CD62L (MEL-14; BioLegend), CD69 (HI.2F3; BioLegend), CD122 (5H4; BioLegend), CD132 (TUGm2; BioLegend), and Vα2 (B20.1; BioLegend). For the staining with antibody specific for CCR7 (4B12; eBioscience), cells were incubated at 37 °C for 30 min before the staining with other antibodies. To isolate naïve CD8+ T cells, spleen cells were stained with biotinylated antibodies specific for CD4, CD44, and B220 (RA3–6B2; BioLegend), and cells that did not bind to the antibodies were enriched with magnetic bead conjugated streptavidin (Miltenyi Biotec). Multicolor flow cytometry and cell sorting were performed on FACSVerse and FACSAriaII (BD Biosciences), respectively.
Cd44 im7
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD44 (IM7) is a monoclonal antibody product from Thermo Fisher Scientific that recognizes the CD44 antigen. CD44 is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b m1 70, by Thermo Fisher Scientific (6 mentions)
Cd62l mel 14, by Thermo Fisher Scientific (5 mentions)
Lsrfortessa, by BD (5 mentions)
Cd4 rm4 5, by Thermo Fisher Scientific (5 mentions)
Facsaria 2, by BD (4 mentions)
Cd8α 53 6, by Thermo Fisher Scientific (3 mentions)
Cd11c hl3, by BD (3 mentions)
Cd11c n418, by Thermo Fisher Scientific (3 mentions)
Cd45 30 f11, by BD (3 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (3 mentions)
F4 80 bm8, by Thermo Fisher Scientific (3 mentions)
Cd4 rm4 5, by BioLegend (3 mentions)
Cd4 gk1, by BD (3 mentions)
Cd8a 53 6, by Thermo Fisher Scientific (3 mentions)
Cd69 h1.2f3, by BD (3 mentions)
Cd45.1 a20, by Thermo Fisher Scientific (3 mentions)
Ifn γ xmg1.2, by BD (3 mentions)
Cd11b m1 70, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Nk1.1 pk136, by Thermo Fisher Scientific (2 mentions)
27 protocols using cd44 im7
1
Murine T Cell Phenotyping by Flow Cytometry
2
Comprehensive Immunophenotyping of Murine Immune Cells
3
Murine Immune Cell Profiling by Flow Cytometry
4
Flow Cytometry Characterization of MSCs
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Fluorescence-activated cell sorting flow cytometry analysis was performed using CD29 (TS2/16; eBioscience, San Diego, CA, United States), CD44 (IM7; eBioscience), CD105 (SN6; eBioscience), CD90 (5E10, eBioscience), CD73 (AD2, eBioscience), CD45 (2D1; eBioscience), CD31 (WM-59; eBioscience), CD34 (4H11; eBioscience), HLA-ABC (W6/32; eBioscience), HLA-DR (L243; eBioscience), C-C chemokine receptor type 1 (CCR1; 5F10B29; BioLegend, San Diego, CA, United States), CCR2 (K036C2; BioLegend), CCR7 (3D12; eBioscience), C-X-C chemokine receptor type 4 (CXCR4; 12G5; eBioscience) and CXCR5 (MU5UBEE; eBioscience) antibodies to confirm that the MSC phenotype was maintained after expansion in the culture. The samples were incubated with antibodies against each surface marker for 30 min, and this treatment was followed by fluorescence-activated cell sorting. Flow cytometry analysis was performed on a LSRFortessa (BD Pharmingen, San Diego, CA, United States).
5
Spleen and Lymph Node Single-Cell Analysis
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Spleen and lymph node samples were forced through 100 µm filters to prepare single-cell suspensions. Dead cells were removed by gradient centrifugation with lymphocyte M (Cedarlane, Burlington, NC, USA) and live cells were stained with surface antibodies for flow cytometric analysis. Data were collected in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Antibodies used: Gr-1 (RB6-8C5, BD Biosciences, San Jose, CA, USA), CD11b (M1/70, eBioscience, San Diego, CA, USA), CD62L (MEL-14, eBioscience, San Diego, CA, USA), CD44 (IM-7, eBioscience, San Diego, CA, USA) and EpCAM (G8.8, eBioscience, San Diego, CA, USA).
6
Multiparametric Flow Cytometry Analysis
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LIVE/DEAD Fixable Red or LIVE/DEAD Fixable Aqua (Invitrogen) staining was performed at 4°C or on ice for 15 min in PBS. Surface Ab staining was performed at 4°C or on ice for 20 min in PBS containing 2% FBS and 2 mM EDTA. For staining intracellular transcription factors, fixation and permeabilization was performed with the eBioscience Intracellular Fixation & Permeabilization Buffer Set Kit prior to intracellular staining for 1 h at 4°C or on ice. Abs for flow cytometry included CD4 (RM4-5; Invitrogen), CD8 (53-6.7; eBioscience), CD25 (PC61; BD Biosciences), CD44 (IM7; eBioscience), CD62L (MEL-14; BD Biosciences), and Foxp3 (FJK-16S; eBioscience). Cells were analyzed or sorted on LSR II, LSRFortessa, and FACSAria II flow cytometers (BD Biosciences) with BD FACSDiva software. Data analysis was performed using FlowJo v.10.2 (Tree Star). FACS data presented in Fig. 3 are gated on forward scatter and side scatter properties indicative of singlet lymphocytes and live CD4+CD8−(CD4SP) cells.
7
Multiparameter Flow Cytometry of Immune Cells
8
Comprehensive Multicolor Flow Cytometry Protocol
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Extracellular staining was preceded by incubation with purified anti-CD16/32 antibodies (FcγRII/III block, 2.4G2) (eBioscience) to block non-specific staining. Cells were stained with FITC-, PE-, PerCP-Cy5.5-, PE-Cy5-, PE-Cy7-, APC-, and APC-H7- labelled appropriate antibodies including: TCRβ (H57-597, eBioscience); CD45 (30-F11, eBioscience and BD Biosciences); CD44 (IM7, eBioscience); CD8α (53–6.7, eBioscience); CD62L (MEL-14, eBioscience); CD4 (GK1.5, BD Biosciences and Miltenyi Biotec); or appropriate isotype Abs. Intranuclear FOXP3 staining was performed using eBioscience PE- or APC-conjugated FOXP3 staining buffer set (FJK-16 s). Intranuclear Ki-67 staining was performed using BD Pharmingen Ki-67 (B56) on permeabilized cells. Six-colour flow cytometry was performed with a FACSCanto cytometer (BD Biosciences). Data files were analysed using FlowJo software (Tree star Inc.).
9
Multiparametric Flow Cytometry Analysis
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Single cell suspensions were incubated with a fixable viability dye (eBioscience) for 20 min at 4°C. Samples were blocked with FC block (24G2 grown in house and mouse serum) for 20 min followed by antibody staining for 20 min. Antibodies used: CD45.1 (A20, eBioscience), CD4 (RM4-5, eBioscience), Va2 (B20.1, BD), MHC II (M5/114.15.2, eBioscience), CD64 (X54-5/7.1, BioLegend), CD8a (53-6.7, eBioscience), CD103 (M290, BD Horizon), Ly6G (1A8 BD), CD69 (H1.2F3, BD), S1PR1 (713412, R&D Systems), interferon-γ (IFN-γ) (XMG1.2, BioLegend), and CD44 (IM7, eBioscience). Samples were washed twice with FACS buffer and acquired on a Miltenyi Macsquant analyzer. Samples were analyzed using FlowJo (Treestar) version 9.7.5.
10
Multiparametric T Cell Analysis
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Thymus and spleen were mechanically teased with glass slides to acquire single cell suspensions and cells were stained with antibodies to the following: CD45.1 (A20, eBioscience), CD45.2 (104, eBioscience), CD4 (RM4‐5, Biolegend), CD8 (53‐6.7, Biolegend), CD25 (PC61.5, Biolegend), CD44 (IM7, eBioscience), Qa2 (695H1‐9‐9, Biolegend), TCRβ (H57‐597, eBioscience), CD69 (H1.2F3, eBioscience), and H‐2Kb (AF6‐88.5, Biolegend). Reagents were conjugated to Pacific Blue, Brilliant Violet (BV) 421, BV510, BV711, PE, PE‐Cy7, PerCP–eFluor 710, allophycocyanin– eFluor 780 and Alexa Fluor 700. Streptavidin PE‐Cy7 (eBioscience) was used to detect biotinylated antibodies. A Foxp3 fixation kit (eBioscience) was used in conjunction with anti‐Foxp3 (FJK‐16s, eBioscience) or the BD Cytofix/Cytoperm Kit (BD Biosciences) to preserve the GFP signal. Data were acquired using a BD LSR Fortessa and FACSDiva 2.6 software and analysed using FloJo software (Tree Star).
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