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Pe cy7 anti cd44 im7

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PE/CY7 anti-CD44 (IM7) is a fluorescently-labeled antibody that binds to the CD44 cell surface glycoprotein. CD44 is a receptor for hyaluronic acid and is involved in cell-cell interactions, cell adhesion, and migration.

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Lab products found in correlation

Rbc lysing buffer, by Merck Group (1 mentions) Mp6 xt22, by BioLegend (1 mentions) 40 m cell strainer, by Greiner (1 mentions) Anti cd69 h1.2f3, by BD (1 mentions) Cytoflex flow cytometer, by Beckman Coulter (1 mentions) Apc cy7 anti tcr β h57 597, by BioLegend (1 mentions) Bv510 anti cd8 53 6.7, by BioLegend (1 mentions) Facscanto, by BD (1 mentions) Quantibrite pe quantification beads, by BD (1 mentions) 7 aad, by BD (1 mentions) Anti cd19 percp 1d3, by BD (1 mentions) Flojo software, by Tree Star (1 mentions) Anti cd4 fitc rm4 5, by BD (1 mentions) Facsverse, by BD (1 mentions) Lsrii flow cytometer, by BD (1 mentions)

4 protocols using pe cy7 anti cd44 im7

1

Multiparametric Flow Cytometry of Immune Cells

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For flow cytometry of tissue culture cells, cells were isolated from mouse spleens and mechanically dissociated with a 40 µm cell strainer (Greiner Bio-One). Red blood cells were lysed with RBC lysing buffer (Sigma). Cells were labeled with aqua live/dead fixable viability dye in PBS (1:500), followed by surface antibodies (1:100) in staining buffer (1% BSA, 1% rat serum in PBS). Antibodies used for surface staining: PE/Dazzle anti-CD4 (RM4-5, Biolegend), APC/CY7 anti-CD8 (YTS156.7.7, Biolegend), PE/CY7 anti-CD44 (IM7, Biolegend), anti-CD69 (H1.2F3, BD Bioscience). For intracellular staining, cells were stained with aqua live/dead dye, followed by surface staining, fixation with perm/fixation solution (Thermofisher), and stained with intracellular antibodies against FITC anti-IFNγ (XMG1.2, Invitrogen) and AF647 anti-tumor necrosis factor α (TNFα) (MP6-XT22, Biolegend) in 1 x perm/wash buffer. Cells were washed and analyzed on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software.
Chen A.C., Xu R., Wang T., Wei J., Yang X.Y., Liu C.X., Lei G., Lyerly H.K., Heiland T, & Hartman Z.C. (2020). HER2-LAMP vaccines effectively traffic to endolysosomal compartments and generate enhanced polyfunctional T cell responses that induce complete tumor regression. Journal for Immunotherapy of Cancer, 8(1), e000258.
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2

Thymocyte Signaling Dynamics

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Thymocytes were incubated with Percp-anti-7AAD (BBIlife sciences), PE-Cy7-anti-CD44 (IM7; Biolegend), APC-Cy7-anti-TCR-β (H57-597; Biolegend), PB-anti-CD4 (RM4-5; Biolegend), and BV510-anti-CD8 (53-6.7; Biolegend) antibodies at 4°C for 30 min and pulsed at 37°C water bath for 5, 15, and 30 min with PMA (50 ng/ml) and Ionomycin (500 ng/ml). The cells were fixed, permeabilized, and stained with phosphorylated FoxO-1 (pFoxO-1, S256; Cell Signaling technology) for 1 h, and further stained with an AF488-Goat anti-rabbit (Life technology) secondary antibody for 30 min.
In order to analyze the phosphorylated level of STAT3, thymocytes were stimulated with PMA and Ionomycin or α-GalCer. For PMA and Ionomycin stimulation, thymocytes were incubated with PMA (50 ng/ml) and Ionomycin (500 ng/ml) in 37°C water bath for 5, 15, and 30 min. For α-GalCer stimulation, thymocytes (2 × 106) were seeded in a 24-well plate in 1640+10% FBS with the addition of α-GalCer (125 ng/ml) for 72 h. PE-anti-STAT3 phospho (pSTAT3, Tyr705; Biolegend) were analyzed. The cells were fixed, permeabilized, and stained with PE-anti-STAT3 phospho (pSTAT3, Tyr705; Biolegend) for 1 h. All flow cytometry data were collected using a FACS Canto (BD Biosciences) and analyzed with FlowJo software. Mean fluorescent intensity is indicated.
Niu L., Xuan X., Wang J., Li L., Yang D., Jing Y., Westerberg L.S, & Liu C. (2018). Akt2 Regulates the Differentiation and Function of NKT17 Cells via FoxO-1-ICOS Axis. Frontiers in Immunology, 9, 1940.
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3

Quantifying TCR Expression Differences

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TCR expression differences were quantified using the anti-TCRβ clone H57–597 PE antibody (eBioscience). Briefly, a few million splenocytes from infected mice were surface stained on ice for 30 minutes with anti-CD11b PerCP Cy5.5 (M1/70; BD), anti-CD11c PerCP Cy5.5 (HL3; BD), anti-CD19 PerCP Cy5.5 (ID3; BD), 7AAD (BD), anti-CD3ε PE CF594 (145–2C11; BD), anti-CD44 PE Cy7 (IM7; Biolegend), anti-CD62L APC Cy7 (MEL-14; BD), anti-CD27 V450 (LG3.A10; BD), anti-CD4 BV510 (RM4–5; Biolegend), anti-PD-1 BV605 (29F.1A12; Biolegend), and anti-CD8 BV785 (53–6.7; Biolegend) antibodies along with the anti-TCRβ antibody. Cells were washed and kept on ice until flow cytometry was carried out using a LSR II (Beckton Dickson). Using the FlowJo software (Tree Star), TCR, CD4 and forward scatter (FSC-A) mean fluorescence intensities (MFIs) of CD4+CD44hi (antigen experienced) and CD4+CD44loCD62+ (naïve) cells was quantified and compared between the two infections per experiment. QuantiBRITE PE quantification beads (BD Biosciences) were used per manufacturer instructions to determine the number of TCRs per cell. For further comparison between infections and across different time points, MFI of antigen experienced cells was normalized to the naïve population within the same sample and quantified as % of naïve ((antigen experienced MFI/naïve MFI) x 100) (39 (link)).
Andargachew R., Martinez R.J., Kolawole E.M, & Evavold B.D. (2018). CD4 T cell affinity diversity is equally maintained during acute and chronic infection Chronic infection fails to alter T cell affinity diversity. Journal of immunology (Baltimore, Md. : 1950), 201(1), 19-30.
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4

Tetramer Staining Assay for T-Cell Epitopes

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MOG42-55-IAg7, hen egg lysozyme (HEL)11-25-IAg7, OVA141-160-IAg7, and human CLIP87-101-IAg7 monomers and tetramers were provided by the National Institute of Health Tetramer Core Facility at Emory University. Mononuclear cells were stained with tetramer immediately post-Percoll gradient isolation. Live cells were recovered from lymphocyte culture by subjecting cells to a Ficoll gradient (Mediatech) and then stained with 4μg/ml tetramer in culture medium for 4h at 37°C. Cells were then stained with anti-CD4-FITC (RM4-5, BD Pharmingen), anti-CD11b-PerCP (M1/70, BD Pharmingen), anti-CD11c-PerCP (HL3, BD Pharmingen), anti-CD19-PerCP (1D3, BD Pharmingen), anti-CD44-PE-Cy7 (IM7, Biolegend), and anti-CD8a-V450 (53-6.1, Tonbo Biosciences) for 30 minutes on ice. Flow cytometry was performed on either a BD Biosciences FACSVerse or LSRII flow cytometer. Data were analyzed using FloJo software (Tree Star).
Kersh A.E., Edwards L.J, & Evavold B.D. (2014). Progression of relapsing-remitting demyelinating disease does not require increased TCR affinity or epitope spread. Journal of immunology (Baltimore, Md. : 1950), 193(9), 4429-4438.
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