LN cells were stained with anti-CD4, anti-CD25, anti-CD44 (IM7; BD), and anti-MHC class II to isolate MHCII−CD4+CD25−CD44− naive CD4+ T cells. Splenocytes were stained with anti-CD11c and anti-MHC class II to isolate MHCII+CD11c+ DCs. Blood cells were stained with anti-MHC class II, anti-CCR3, and anti–Siglec-F, and bone marrow cells were stained with anti–MHC class II, anti-CD11b, and anti–Siglec-F to isolate MHCII−CCR3highSiglec-Fhigh and MHCII−CD11bintSiglec-Fhigh eosinophils, respectively. Small intestinal epithelial cell preparation was stained with anti-CD45 (30-F11; eBioscience) and anti-EpCAM (G8.8; BioLegend) to isolate CD45−EpCAM+ intestinal epithelial cells. Small intestinal LP cells were stained with anti-CD4, anti-CD25, anti–MHC class II, and anti–Siglec-F; MHCII−Siglec-F+ eosinophils and MHCII−Siglec-F−CD4+CD25− T cells were sorted on a MoFlo Astrios or MoFlo XDP (Beckman Coulter).
Anti cd44 im7
Manufactured by BD
Sourced in United States
Anti-CD44 (IM7) is a monoclonal antibody that specifically binds to the CD44 cell surface antigen. CD44 is a transmembrane glycoprotein involved in cell-cell and cell-matrix interactions. The Anti-CD44 (IM7) antibody can be used for the identification and characterization of CD44-expressing cells in various applications.
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Lab products found in correlation
Anti cd45 30 f11, by BD (3 mentions)
Anti cd62l mel 14, by BioLegend (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti cd69 h1.2f3, by Thermo Fisher Scientific (2 mentions)
Anti cd25 pc61, by BD (2 mentions)
Anti cd4 rm4 5, by Thermo Fisher Scientific (2 mentions)
Anti cd8a 53 6, by BioLegend (2 mentions)
Anti cd62l mel 14, by BD (2 mentions)
Anti b220 ra3 6b2, by BD (2 mentions)
Anti cd11b m1 70, by BD (2 mentions)
Diva software, by BD (2 mentions)
Sytox blue, by Thermo Fisher Scientific (2 mentions)
Lsrii system, by BD (2 mentions)
Anti cd19 6d5, by BioLegend (2 mentions)
Anti tcrγδ gl3, by BioLegend (2 mentions)
Anti cd45 30 f11, by Thermo Fisher Scientific (1 mentions)
Moflo astrio, by Beckman Coulter (1 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
Anti cd4, by BD (1 mentions)
Anti epcam g8.8, by BioLegend (1 mentions)
13 protocols using anti cd44 im7
1
Isolation of Immune Cell Subsets
2
Flow Cytometric Analysis of Lymphocytes
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Flow cytometric analyses were done at the Robert H. Lurie Flow Cytometry Core Facility (RHLCCC) using the Fortessa (BD) flow cytometry system. For surface staining, cells were incubated with fluorochrome-conjugated antibodies for 30 minutes at 4 °C using 1:200 dilutions of each antibody (unless otherwise specified). Cells were washed twice in FACS buffer prior to being analyzed or stained intracellularly using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Dead cells were excluded either using DAPI (Life technologies) or LIVE/DEAD® Fixable Dead Cell Stain Kits (Life technologies). Forward and side scatter was used to identify live lymphocytes. Data analysis was performed using FlowJo (version 9.6.2) software (Tree Star). Antibodies against mouse CD4 (GK1.5), anti-CD8α (53–6.7), anti-CD62L (MEL-14), anti-GATA3 (L50–823) and anti-CD44 (IM7) were from BD Biosciences. An antibody against mouse CD25 (PC61) was from BioLegend. Antibodies against mouse GITR (DTA-1), anti-CTLA4 (UC10–4B9), anti-Foxp3 (FJK-165), anti-IFNγ (XMG1.2), anti-IL17A (eBio17B7), anti-IL4 (11B11), anti-RORγt (B2D), anti-T-bet (eBio4B10) and anti-CD69 (H1.2F3) were from eBioscience. An antibody against mouse Neuropilin-1 was from R&D Systems. CellTrace™ CFSE Cell Proliferation Kit (Thermo Scientific) for flow cytometry was used to track cell proliferation in in vitro Treg suppression assays.
3
Characterizing OT-I CTL Activation Markers
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Expression of activation‐associated surface molecules on OT‐I CTLs was evaluated on day 3 or 4 post‐transduction or the day that adoptive transfer was performed, which was usually day 6 post‐isolation. Cells were stained with anti‐CD8α (53–6.7, BD Biosciences, San Jose, CA, USA), anti‐CD44 (IM7, BD Biosciences), anti‐CD25 (PC61, BD Biosciences), anti‐CD69 (H1.2F3, BD Biosciences), anti‐CD62L (MEL‐14, eBioscience, Vienna, Austria) and anti‐Vα2 (B20.1, BD Biosciences). Antibodies were used at 1 μg mL−1 in running buffer (5% fetal bovine serum and 2 mm ethylenediaminetetraacetic acid, 0.01% sodium azide in 1 × phosphate‐buffered saline) and the cells were stained for 20 min at 4°C prior to washing two times with running buffer. Cell viability was evaluated using 0.5 μg mL−1 4,6‐diamidino‐2‐phenylindole (Thermo Fisher Scientific) or the LIVE/DEAD Fixable near‐IR (Thermo Fisher Scientific). Data were collected on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For fluorescence‐activated cell sorting of medium to high mCherry expressing CTL population, cells were resuspended in FACS buffer (5% fetal calf serum, 2 mm ethylenediaminetetraacetic acid in 1 × phosphate‐buffered saline) and incubated with 4,6‐diamidino‐2‐phenylindole. Cells were either used fresh or cryopreserved according to established methods.85
4
Multiparametric Flow Cytometry Analysis
5
Murine Splenic Immune Cell Analysis
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Spleens were aseptically isolated from ten mice from each group 4 weeks after challenge. Splenocyte suspensions were prepared in RPMI-1640 culture media supplemented with 10% FBS, after RBC lysis with red blood cell lysing buffer hybrid-max (Sigma-Aldrich, St. Louis, MO, USA), for flow cytometry analysis, antibody secreting cell (ASC) assays and cytokine analysis. Cells were stained with trypan blue (Welgene, Daegu, South Korea) and counted with a hemocytometer chamber under a microscope. Splenocytes from each animal were resuspended in staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1 M PBS). Cells from individual mouse were separately incubated with Fc Block (clone 2.4G2; BD Biosciences, CA, USA) to block non-specific binding at 4°C for 15 min, and then stained at 4°C for 30 min with different combinations of FITC, PE, PE-Cy5, PE-Cy7 or APC conjugated anti-CD3e (145-2c11), anti-CD4 (GK1.5), anti-CD8a (53–6.7) (BD Biosciences, CA, USA). For memory T cell responses, anti-CD44 (IM7) and anti-CD62L (MEL-14) were used (BD Biosciences, CA, USA) as indicated [28 ]. For memory B cell responses, anti-CD45R/B220 (RA3-6B2), anti-CD27 (LG-3A10) and anti-IgG1 (A85-1) were used (BD Biosciences, CA, USA) [29 (link)]. Events were acquired on BD Accuri C6 Flow Cytometer (BD Biosciences, CA, USA) and data were analyzed using C6 Analysis software (BD Biosciences, CA, USA).
6
Multiparametric Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells
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Anti-CD3 (17A2, cat. 561388), anti-CD4 (RM4-5, cat. 553047), anti-CD44 (IM7, cat. 561859), anti-CD62L (MEL-14, cat. 560516), anti-B220 (RA3-6B2, cat. 553091), anti-Foxp3 (MF23, cat. 562996) and anti-CD23 (B3B4, cat. 561772) were purchased from BD company (Franklin Lakes, NJ, USA). Anti-CD8a (53–6.7, cat. 100734), anti-IgM (RMM-1, cat. 406531) and anti-CD21 (7E9, cat. 123411) were obtained from BioLegend (San Diego, CA, USA). For flow cytometry experiments, total eight axillary LNs were dissected from four each of normal and tumor-bearing mice and pooled separately. Cells were harvested and washed with 1X PBS. Cells were stained with antibodies within 1:100 dilution for 40 min at 4°C with gentle mix, then washed with 1X PBS prior to Attune NxT cytometer (Thermo Fisher Scientific) analysis. For Foxp3 staining, cells were mixed with 1 ml of Fix/Perm solution and incubated at room temperature for 15 min. Cells were washed with 1X PBS and stained with anti-Foxp3 antibody for flow cytometry analysis.
7
Multiparameter Flow Cytometry Analysis
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Cells were washed with PBS containing 0.5% BSA and incubated for 30 minutes on ice with the following antibodies: anti-CD4 (RM-4.5; eBioscience), anti-CD8 (53.6.7; BD), anti-CD44 (IM.7; BD), anti-CD25 (PC61; BD), anti-CD62L (MEL14; Biolegend), anti-TCRβ (H57-597; eBioscience), anti-TCRγδ (BD), anti-CD69 (H1.2F3; eBioscience), anti-CD11b (M1/70; BD), anti-Gr1 (RB6-8C5; eBioscience) and anti-HSA (M1/69, eBioscience). Cells were then washed in PBS with 0.5% BSA and data was collected using a Fortessa cytometer (BD Bioscience) and analyzed using FlowJo software (Treestar, Ashland, OR).
In the case of intracellular staining, cells were fixed and permeabilized using the Foxp3 buffer staining kit (eBioscience) according to the manufacturer’s instructions prior to staining for intracellular Foxp3 expression using an anti-Foxp3 antibody (FJK-16s, BD), for 30 minutes.
For Annexin V staining, cells were washed with PBS and stained with BD Pharmingen Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.
The detection of BrdU was performed using BD Pharmingen BrdU Flow Kit according to the manufacturer’s instructions.
In the case of intracellular staining, cells were fixed and permeabilized using the Foxp3 buffer staining kit (eBioscience) according to the manufacturer’s instructions prior to staining for intracellular Foxp3 expression using an anti-Foxp3 antibody (FJK-16s, BD), for 30 minutes.
For Annexin V staining, cells were washed with PBS and stained with BD Pharmingen Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.
The detection of BrdU was performed using BD Pharmingen BrdU Flow Kit according to the manufacturer’s instructions.
8
Phenotype Analysis of Immune Cells
9
Multiparameter Flow Cytometry Analysis
10
Immune Cell Profiling of Tumor Samples
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