Splenocytes were either stimulated with survivin or MUC1 protein for 12 h. Following incubation, the cells were stained with the following surface antibodies: APC anti-mouse CD8a (Biolegend), PE-Cy7 anti-mouse CD4 (Biolegend). Cells were then permeabilized and washed with the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer’s instructions. Intracellular Ki67 were subsequently stained with PE anti-mouse Ki67 (Biolegend). For central memory T cells (TCM) and effector memory T (TEM) cells, in addition to APC anti-mouse CD8a (Biolegend), PE anti-mouse CD44 (Biolegend) and PE-Cy7 anti-mouse CD62L (Biolegend) were used for cell surface staining. After staining, cells were washed, fixed, and analyzed using flow cytometry.
Pe anti mouse cd44
Manufactured by BioLegend
Sourced in United States
PE anti-mouse CD44 is a monoclonal antibody that binds to the mouse CD44 cell surface antigen. CD44 is a glycoprotein involved in cell-cell interactions, cell adhesion, and migration. This antibody can be used for the identification and characterization of mouse CD44-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Pe anti mouse cd86, by BioLegend (2 mentions)
Fitc anti mouse cd11c, by BioLegend (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Pe anti mouse cd45, by BioLegend (2 mentions)
Pe anti mouse sca 1, by BioLegend (2 mentions)
Apc anti mouse cd62l, by BioLegend (2 mentions)
Pe cy7 anti mouse cd62l, by BioLegend (1 mentions)
Apc anti mouse cd8a, by BioLegend (1 mentions)
Pe cy7 anti mouse cd4, by BioLegend (1 mentions)
Pe anti mouse ki67, by BioLegend (1 mentions)
Apc anti mouse cd34, by BioLegend (1 mentions)
Apc anti mouse cd117, by BioLegend (1 mentions)
Apc anti mouse cd31, by BioLegend (1 mentions)
Fc receptor blocking agent, by BioLegend (1 mentions)
Pe anti mouse cd90, by BioLegend (1 mentions)
Mk 801, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Apc anti mouse cd45, by BioLegend (1 mentions)
Fitc anti mouse cd3, by BioLegend (1 mentions)
Pe cy7 anti mouse cd45, by BioLegend (1 mentions)
7 protocols using pe anti mouse cd44
1
Multicolor Flow Cytometry Analysis of T Cell Subsets
2
Characterization of CXCR4 Expression in MSCs
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MSCs were detected the expression of specific surface markers, including PE anti-mouse Sca-1, PE anti-mouse CD90.2, PE anti-mouse CD29, PE anti-mouse CD44, PE anti-mouse CD45, APC anti-mouse CD31, APC anti-mouse CD34, and APC anti-mouse CD117 (Biolegend, San Diego, CA).
After MSCs were treated with 3 mM NMDA (Sigma-Aldrich) or 50 μM MK801 (Sigma-Aldrich) for 24 h, APC anti-mouse C-X-C chemokine receptor type 4 (CXCR4; Biolegend) was used for surface staining to study the effect of NMDA receptor activation on CXCR4 expression in MSCs.
MSCs were harvested and then blocked with Fc receptor blocking agent (BioLegend) for 10 min on ice. The antibodies were added and incubated for 30 min at 4°C in dark. The cells were then washed with 0.1% BSA and fixed with 1% paraformaldehyde in PBS. Cell surface staining was analyzed using a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ) within 24 h of staining. FlowJo software version 7.6.1 (FlowJo, Ashland, OR) was used for data analyses.
After MSCs were treated with 3 mM NMDA (Sigma-Aldrich) or 50 μM MK801 (Sigma-Aldrich) for 24 h, APC anti-mouse C-X-C chemokine receptor type 4 (CXCR4; Biolegend) was used for surface staining to study the effect of NMDA receptor activation on CXCR4 expression in MSCs.
MSCs were harvested and then blocked with Fc receptor blocking agent (BioLegend) for 10 min on ice. The antibodies were added and incubated for 30 min at 4°C in dark. The cells were then washed with 0.1% BSA and fixed with 1% paraformaldehyde in PBS. Cell surface staining was analyzed using a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ) within 24 h of staining. FlowJo software version 7.6.1 (FlowJo, Ashland, OR) was used for data analyses.
3
Radioisotope Production and Cellular Analysis
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The radioisotope 211At was produced via 209Bi (α, 2n) 211At reaction through CS-30 cyclotron according to the published protocol [25 (link)]. Bovine serum albumin (BSA), Manganese chloride tetrahydrate (MnCl2·4H2O), and sodium hydroxide (NaOH) were from Sigma-Aldrich. Roswell park memorial institute (RPMI) 1640 medium, penicillin-streptomycin, and fetal bovine serum (FBS) were obtained from Gbico. The cell counting kit-8 (CCK-8) was obtained from Biosharp. The anti-mouse PD-L1 antibody was obtained from BioXcell (clone:10F.9G2). APC anti-mouse CD45(Catalog: 103111), FITC anti-mouse CD3 (Catalog: 100203), BV421™ anti mouse CD4(Catalog: 100437), APC/Cy7 anti-mouse CD8a (Catalog: 100714), PE anti-mouse FOXP3 (Catalog: 126403), FITC anti-mouse CD11c (Catalog: 117305), PE anti-mouse CD86 (Catalog:105007), APC anti-mouse CD80 (Catalog: 104713), PE/Cy7 anti-mouse CD45 (Catalog: 103113), PE anti-mouse CD44(Catalog:103023) and BV421- anti-mouse CD62L (Catalog: 104435) antibodies were obtained from Biolegend, Tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) ELISA kit was purchased from Multisciences Biotech, Co., Ltd.
4
Investigating Immune Modulation with CpG ODN, Anti-OX40, and Anti-PD-1
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CpG ODN 2359 was obtained from Tsingke Biological Technology Co. (Beijing, China). Anti-OX40 agonistic antibody (αOX40, Clone OX-86) was provided by GenScript USA Inc. Murine anti-PD-1 antibody (G4C2) was provided by Shanghai Junshi Biosciences Co.,Ltd (Suzhou, China).Monoclonal antibodies (mAbs) used for flow cytometry were listed as flow: FITC anti-mouse CD45 (Biolegend, USA), PE/Dazzle™ anti-mouse CD3 (Biolegend, USA), FITC anti-mouse CD8 (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse CD4 (Biolegend, USA), FITC anti-mouse CD11c (Biolegend, USA), PE anti-mouse CD86 (Biolegend, USA), PE/Cyanine7 anti-mouse CD80 (Biolegend, USA), PE anti-mouse CD44 (Biolegend, USA), PE/Cyanine7 anti-mouse CD62L (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse PD-1 (Biolegend, USA), PE/Cyanine7 anti-mouse OX40 (Biolegend, USA), FITC anti-mouse CD11b (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse F4/80 (Biolegend, USA), APC anti-mouse CD206 (Biolegend, USA), Mouse Regulatory T cell staining kit (eBioscience, USA).
5
Multicolor Flow Cytometry Panel
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Antibodies used include: anti-mouse CD45 APCCY7 (Clone 30-F11 Biolegend Cat #103116) and anti-mouse CD45 Eflour 450 (Clone 30-F11 Invitrogen Cat#48-0451-82); anti-mouse LY6G (Clone 1A8; Biolegend Cat#127623); anti-Mouse CD3 Pacific Blue (Clone 145-2C11 Biolegend Cat#100334); anti-Mouse CD3e, PerCP-Cy5.5 (Clone: 145-2C11, eBioscience, Cat#: 45-0031-82); anti-Mouse CD8a PerCP (Clone Ly-2 BDBiosciences Cat#M037858); anti-Mouse CD4, FITC (Clone: GK1.5; eBioscience, Cat#: 25-0041-82); anti-mouse CD19 PE (Clone eBio1D3 ThermoFisher Cat# 12-0193-83); anti-mouse CD11c APC (Clone N418; Biolegen Cat #117310); anti-mouse CD11b FITC (Clone M1/70.15 Invitrogen Cat #RM2801); anti-mouse CD25 PE (Clone 3C7 Biolegend Cat#101904); anti-mouse CD44 PE (Clone 1M7; Biolegend Cat# 10,008); anti-mouse CD62L APC (Clone Mel-14 Biolegend Cat# 104411).
6
Multiparameter Flow Cytometry Analysis
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Single-cell suspensions from mouse tissues were prepared in PBS containing 2% FBS. Cells were fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) or FoxP3/Transcription Factor Staining Buffer Set (eBioscience). For surface marker analysis, cells were stained with the indicated antibodies (Abs) in PBS containing 2% FBS. Intracellular staining was performed according to the manufacturer's instructions. The following antibodies were used: anti-mouse CD4 APC-cy7, anti-mouse CD25 BV421/PE, anti-mouse FoxP3 APC, anti-mouse CD62L APC, anti-mouse CD44 PE, anti-mouse IFN-γ PE, anti-mouse IL-17A APC, anti-CD4 APC, and anti-Foxp3 PE from BioLegend. The anti-mouse/rat Ki67 was purchased from ebioscience. The anti-Akt-pSer473 rabbit antibody and anti-Akt-pThr308 rabbit antibody were purchased from Cell Signaling Technology as primary antibodies, and the donkey anti-rabbit AF594 secondary antibody was purchased from Invitrogen. Finally, the cells were washed, resuspended, and analyzed with a BD LSRFortessa X-20 flow cytometer.
7
Flow Cytometric Profiling of Murine MSCs
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For flow cytometric analysis, MSC were stained with the following antibodies: anti-mouse Sca1PE, anti-mouse CD73PE/Cy7, anti-mouse CD117APC (c-kit), anti-mouse CD45PE, anti-mouse CD44PE, anti-mouse CD95PE (Biolegend, San Diego, CA, USA), goat anti-mouse CD105 and the secondary rabbit anti-goatFITC (Invitrogen, Carlsbad, CA, USA). The antibody mix that was used to label the splenocytes contained anti-mouse CD45PE, anti-mouse CD4BV784 and anti-mouse CD8aAPCfire (Biolegend, San Diego, CA, USA). FasL was detected using anti-FasLAF647 antibody (MLF4 clone, Bio-Rad, CA, USA). For all the antibodies, we used the corresponding isotypes from the same sources. Samples were analyzed using a Cytoflex flow cytometer (Beckman Coulter, Indianapolis, IN, USA) and CytExpert software. Determination of the proliferation index of the splenocytes was done based on the carboxyfluorescein succinimidyl ester (CFSE) readings that were processed using ModFit LTTM software (Verity Software House, Topsham, ME, USA). For the CFSE analysis, the unstimulated splenocytes were used as the parent (basal fluorescence).
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