Tumor tissues were digested both mechanically by chopping with razor blades and chemically with 1mg/mL type IA collagenase (Sigma-Aldrich) for 30 minutes at 37°C. Following digestion, cell suspensions were washed, filtered and stained as previously described (O’Sullivan et al., 2012 (link)). The following antibodies were used: Ly6C (ER-MP20, Serotec), MHCII (M5/114 15.2, eBioscience), Ly6G (1A8, Biolegend), CD8 (53-6.7, eBioscience), CD44 (IM7, Biolegend), CD3 (17A.2, Biolegend), CD4 (GK1.5, Biolegend), CD69 (H1.2F3, Biolegend), Granzyme B (NGZB, eBioscience), IFNγ (XMG 1.2, Biolegend), TCRβ (H57-597, Biolegend), B220 (RA3-6B2, eBioscience), NK1.1 (PK136, Biolegend), CD11b (M1/70, eBioscience), CD45 (30-F11, Biolegend). Stained cell suspensions were analyzed on a BD FACS CANTO II (BD Biosciences).
Cd44 im7
Manufactured by BioLegend
Sourced in United States
CD44 (IM7) is a monoclonal antibody 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
Cd62l mel 14, by BioLegend (10 mentions)
Cd4 gk1, by BioLegend (6 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (6 mentions)
Cd45.1 a20, by BioLegend (6 mentions)
Cd45.2 104, by BioLegend (6 mentions)
Cd8 53 6, by BioLegend (5 mentions)
Ki67 sola15, by Thermo Fisher Scientific (4 mentions)
Cd69 h1.2f3, by BioLegend (3 mentions)
Cd45 30 f11, by BioLegend (3 mentions)
Icos c398.4a, by BioLegend (3 mentions)
Cd25 7d4, by BD (3 mentions)
Il 17a tc11 18h10, by BD (3 mentions)
Nrp1 fab566a, by R&D Systems (3 mentions)
Ctla4 14d3, by Thermo Fisher Scientific (3 mentions)
Gitr dta 1, by Thermo Fisher Scientific (3 mentions)
Cd4 h129, by BD (3 mentions)
Cd62l mel 14, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Cd3 17a2, by BioLegend (2 mentions)
Cd11b m1 70, by Thermo Fisher Scientific (2 mentions)
29 protocols using cd44 im7
1
Multiparameter Flow Cytometry of Tumor Cells
2
Activation-induced T Cell Immunophenotyping
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LN single cell suspensions were stained using the fixable viability dye ZombieRed (Biolegend) for 15 min. at room temperature, and added for 30 minutes at 37°C to tissue culture plates pre-coated overnight with αCD3ε (clone 145–2C11) and αCD28 (clone 37.51) antibodies (at 10 μg/ml of each antibody), or to uncoated control plates. Samples were then fixed in 4% paraformaldehyde (PFA) for 10 minutes at room temperature, and permeabilized for 20 minutes through dropwise addition of 1 ml ice-cold methanol. Cells were then stained for CD90.2 (30-H12), CD4 (GK1.5), CD8α (53–6.7), CD44 (IM7) (BioLegend), Foxp3 (FJK-16s, eBioscience), pFoxo1 (Thr24)/Foxo3a (Thr32), p-c-Jun (Ser73) (D47G9) (Cell Signaling) and GFP (rabbit polyclonal Ab) (Invitrogen).
3
Multiparametric flow cytometry for immune cells
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Samples were blocked with 2% normal mouse serum. Fixable yellow (L34959, Invitrogen, Carlsbad, CA, USA) or 7AAD (Cat 00699350, eBioscience, Waltham, MA, USA) was used to stain live/dead cells. Anti-mouse antibodies used were CD3 (17A2, eBioscience), CD4 (GK1.5, BioLegend, San Diego, CA, USA), TCR Vα2 (KB5-C20, BD Pharmingen), CD8a (53-6.7, BioLegend), CD44 (IM7, Biolegend), CD62L (MEL-14, eBioscience), H-2Db (KH95, BD Pharmingen), H-2Kb (AF6–88.5, BD Pharmingen), PD-L1 (MIH5, eBioscience). Anti-human antibodies used were HLA ABC (W6/32, Biolegend), PD-L1 (29E.283, Biolegend), anti-mouse IgG2a K Isotype (Biolegend), and anti-mouse IgG2b K (Biolegend).
Fluorescence was measured on BD LSR Fortessa X-20 or BD FACSVerse flow cytometer (BD Biosciences, North Ryde, NSW, Australia) and data analyzed using the FlowJo, LLC software (BD Biosciences, Franklin Lakes, NJ, USA).
Fluorescence was measured on BD LSR Fortessa X-20 or BD FACSVerse flow cytometer (BD Biosciences, North Ryde, NSW, Australia) and data analyzed using the FlowJo, LLC software (BD Biosciences, Franklin Lakes, NJ, USA).
4
Phosphorylation Analysis of ERK1/2 and AKT in PBMCs
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To evaluate phosphorylation of ERK1/2, PBMCs were stimulated with phorbol-12-myristate-13-acetate (PMA) and ionomycin (1 ng/mL:1 mM; Sigma-Aldrich) for 5 minutes in RPMI 1640 supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). Cells were then fixed and permeabilized with FIX & PERM A/B buffers and stained for intracellular phosphorylated ERK1/2 (pERK1/2) and surface antigens. pERK1/2 was stained with rabbit anti-pERK1/2 (Cell Signaling Technology, Danvers, MA) and Alexa 647–conjugated donkey anti-rabbit immunoglobulin G (IgG) (Thermo Fisher Scientific). To evaluate phosphorylation of AKT, PBMCs were stimulated with insulin (Wako Pure Chemical Industries) for 10 minutes. Rabbit anti-phosphorylated AKT (pAKT) antibodies (Cell Signaling Technology) were used. The following antibodies were also used: anti-human CD4 (SK3), CD8 (SK1), CD20 (L27), CD23 (M-L233), CD68 (Y1/82A), and CD69 (FN50); and anti-mouse CD4 (RM4-5), CD19 (1D3), CD62L (MEL-14) (Becton Dickinson, Franklin Lakes, NJ), and CD44 (IM7; BioLegend). Cells were analyzed by using a BD FACSLyric instrument (Becton Dickinson, Franklin Lakes, NJ) with FlowJo software, version 10.4 (Becton Dickinson).
5
Multiparameter FACS and Cell Sorting
6
Flow Cytometry Analysis of OT-II Cells and DCs
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The following antibodies and reagents were used: anti-CD4 (RM4-5; Biolegend/eBioscience), -CD11c (N418; BD/Biolegend), -CD44 (IM7; Biolegend), -CD45.1 (A20; Biolegend), -CD62L (MEL-14; Biolegend), CD127 (A7R34; BD/Biolegend), -KLRG-1 (2F1; Biolegend/eBioscience), -Ki67 (SolA15; eBioscience), -I-Ab (AF6-120.1; Biolegend), -Thy1.1 (OX-7; Biolegend), -TCR Vα2 (B20.1; Biolegend), streptavidin-BV510 (Biolegend) and streptavidin-PE (Biolegend). For intranuclear staining of Ki67, cells were first stained with the indicated antibodies directed against cell surface molecules. Afterwards cells were fixed with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions and subsequently incubated with anti-Ki67 for 30 min at 4°C. Samples were measured on LSRFortessa flow cytometer (Becton Dickinson) and analyzed by FlowJo 9 and 10 software (FlowJo, LLC). To calculate the fold expansion of OT-II cells or DCs, the respective cell populations were quantified. For each experiment a mean value was calculated for the RagWT group. Finally, cell numbers of individual mice, including RagWT mice, were calculated in relation to the mean value of the RagWT group. Relative mean fluorescence intensities (MFIs) and relative frequencies of OT-II cells or DCs were calculated in analogy.
7
Comprehensive Immune Cell Profiling
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Cells from the thymus, spleen or lymph nodes were resuspended in staining buffer at a density of 2 × 106 cells/ml. The cells were incubated with anti-FcγRII/III monoclonal antibody (mAb) (2.4G2), followed by antibodies against CD4 (RM4-5, BD Pharmingen), CD8 (53–6.7, BD Pharmingen), B220 (RA3-6B2, BioLegend), Thy1.1 (53–2.1, BioLegend), Thy1.2 (OX-7, BioLegend), CD44 (IM7, BioLegend), CD62L (MEL-14, BioLegend), Dll1 (HMD1-5, eBiosciences), Dll4 (HMD4-1, eBiosciences), Jagged1 (HMJ1-29, eBiosciences) or Jagged2 (HMJ2-1, eBiosciences). After gating out cells that were positive for 7-AAD, excluding the doublets, the fluorescence intensity of 105 cells was measured with a FACS Canto II flow cytometer (BD Biosciences, CA) and analyzed with FACSDiva (BD Biosciences) or FlowJo (Tree Star) software programs. For intracellular staining, cells were fixed with an intracellular staining kit (72–5775, eBioscience), permeabilized and stained with Foxp3 (3G3, Tonbo Biosciences) antibody.
8
Comprehensive Immune Cell Profiling
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Cells were stained with fixable LIVE/DEAD dye (Life Technologies), blocked with anti-CD16/32, and stained with antibodies for CD45 (30-F11; BioLegend), CD3 (17A2; BioLegend), CD4 (GK1.2; eBiosciences), CD11b (M1/70; BioLegend), CD11c (N418), CXCR3 (CXCR3–173), Ly6C (HK1.4; BioLegend), CD44 (IM7; BioLegend), CD62L (MEL-14; Invitrogen), and/or major histocompatibility complex class II (M5/114.15.2; eBiosciences). For IFN-γ production, the cells were stimulated for 6 hours with phorbol myristate acetate and ionomycin in the presence of brefeldin A and monensin for the final 4 hours. The cells were stained for extracellular markers and then fixed with fixation/permeabilization buffer, and intracellular staining for Foxp3 and IFN-γ was performed in permeabilization buffer. Fluorescence minus one controls, single color controls, and an unstained control were used for all experiments. Data were collected on an LSRFortessa (BD) and were analyzed using FlowJo (BD). Microglia are defined as CD45intCD11b+, while inflammatory monocytes are CD45hiCD11b+Ly6c+.
9
Recombinant pcDNA3.1(-)-dsNKG2D-IL-21 Plasmid
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The recombinant pcDNA3.1(−)–dsNKG2D–IL-21 plasmid was previously generated in our laboratory in accordance with standard methods.10 The gene fragment of β2-microglobulin signal peptides was also added upstream the NKG2D gene. Liposome (Lipofectamine™ 2000) was obtained from Invitrogen (Grand Island, NY, USA). EndoFree Plasmid Maxi Kit from Qiagen (Düsseldorf, Germany) was further used to deplete endotoxin. Chitosan (MW 20 kDa) with 75% to 85% deacetylation was purchased from Yuhuan County Marine Chemical Company (Yuhuan, Zhejiang, China). Antibodies against CD4 (GK1.5), CD8 (53.67), CD49b (DX5), CD69 (LG.3A10), CD107a (1D4B), and CD44 (IM7) were all purchased from Biolegend (San Diego, CA, USA). Recombinant IL-21 and murine NKG2D-Ig proteins were from R&D systems (Minneapolis, MN, USA). YAC-1, NIH-3T3, CT-26, and RAW264.7 cell lines were obtained from ATCC.
10
Multiparametric Flow Cytometry Analysis
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Cell suspensions from mouse liver, BM (one femur and tibia), spleen, thymus, mesenteric lymph nodes, and peritoneal cavity were processed as previously described (47 (link)), and data were acquired on a FACSCanto10c (BD) and analyzed with FlowJo Software (Tree Star).
The following anti-mouse antibodies were used: B220 (RA3-6B2, BioLegend), BAFFR (7H22-E16,BD), BP-1 (6C3, BioLegend), CD3e (145-2C11, eBioscience), CD4 (GK1.5, eBioscience), CD5 (53-7.3, BioLegend), CD8 (53-6.7, BioLegend), CD9 (MZ3, BioLegend), CD11b (M1/70, eBioscience), CD19 (6D5, 1D3, BioLegend), CD21/CD35 (7E9, BioLegend), CD23 (B3B4, BioLegend), CD24 (M1/69, BioLegend), CD25 (PC61, Biolgend), CD43 (S7, BioLegend), CD44 (IM7, BioLegend), CD45.1 (A20, BioLegend), CD45.2 (104, BioLegend), CD69 (H1.2F3, BioLegend), CD80 (16-10A1, BioLegend), CD86 (GL1, BioLegend), CD93 (AA4.1, BioLegend), DAPI (BIOTIUM), Gr-1 (RB6-8C5, eBioscience), IgDa (AMS-9.1, BioLegend), IgD (11-26c, BioLegend), IgMa (DS-1, MA-69, BioLegend), IgM (Il/41, RMM-1, BioLegend), TCRγδ (GL3, BioLegend), Live dead dye (Zombie Aqua Dye, BioLegend), Live dead dye (Zombie NIR, BioLegend), pERK (4B11B69, BD), pPLCγ2 (K86-1161, BD), Lin28b (AP1485C, ABGENT), and anti-rabbit IgG (BioLegend).
The following anti-mouse antibodies were used: B220 (RA3-6B2, BioLegend), BAFFR (7H22-E16,BD), BP-1 (6C3, BioLegend), CD3e (145-2C11, eBioscience), CD4 (GK1.5, eBioscience), CD5 (53-7.3, BioLegend), CD8 (53-6.7, BioLegend), CD9 (MZ3, BioLegend), CD11b (M1/70, eBioscience), CD19 (6D5, 1D3, BioLegend), CD21/CD35 (7E9, BioLegend), CD23 (B3B4, BioLegend), CD24 (M1/69, BioLegend), CD25 (PC61, Biolgend), CD43 (S7, BioLegend), CD44 (IM7, BioLegend), CD45.1 (A20, BioLegend), CD45.2 (104, BioLegend), CD69 (H1.2F3, BioLegend), CD80 (16-10A1, BioLegend), CD86 (GL1, BioLegend), CD93 (AA4.1, BioLegend), DAPI (BIOTIUM), Gr-1 (RB6-8C5, eBioscience), IgDa (AMS-9.1, BioLegend), IgD (11-26c, BioLegend), IgMa (DS-1, MA-69, BioLegend), IgM (Il/41, RMM-1, BioLegend), TCRγδ (GL3, BioLegend), Live dead dye (Zombie Aqua Dye, BioLegend), Live dead dye (Zombie NIR, BioLegend), pERK (4B11B69, BD), pPLCγ2 (K86-1161, BD), Lin28b (AP1485C, ABGENT), and anti-rabbit IgG (BioLegend).
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