C57BL/6 J (CD45.2) and B6.SJL-PtprcaPepcb/BoyJ mice (B6.SJL, CD45.1) were provided by the Animal Centre of the Institute of Hematology & Blood Diseases Hospital, CAMS & PUMC. Sex- and age-matched mice were used and maintained in the SPF-certified animal facility in the same center. The procedures for the animal experiments were approved by the Animal Care and Use Committee at the institutions involved in this study. After transplantation, the mice were raised in sterile squirrel cages in a positive pressure room. All of the antibodies used in this paper were obtained from eBioscience (San Diego, CA, USA), including PE-, FITC-, or PerCP-Cy5.5-conjugated anti-CD45.1, PE-conjugated anti-CD45.2, PE-conjugated anti-Sca-1, APC-conjugated anti-c-Kit, PE-conjugated anti-CD3, PE-Cy7-conjugated anti-B220, APC-conjugated anti-Mac-1, biotin-conjugated lineage markers (anti-CD3, −CD4, −CD8, −B220, −CD11b, −Gr-1, and -Ter119) and streptavidin-PE-Cy7. Neutralizing antibodies against MIP-3β and CCR7 were purchased from R&D Systems (Minneapolis, MN, USA).
Pe conjugated anti cd3
Manufactured by Thermo Fisher Scientific
Sourced in United States
PE-conjugated anti-CD3 is a monoclonal antibody that binds to the CD3 complex on the surface of T cells. The antibody is conjugated with the fluorescent dye phycoerythrin (PE), which allows for the detection and analysis of CD3-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Pe cy7 conjugated anti b220, by Thermo Fisher Scientific (1 mentions)
Percp cy5 5 conjugated anti cd45, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti c kit, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti sca 1, by Thermo Fisher Scientific (1 mentions)
Perm wash solution, by BD (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ mab, by Thermo Fisher Scientific (1 mentions)
Facs fortessa flow cytometer, by BD (1 mentions)
Apc conjugated anti cd4, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti gr 1, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd19, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti f4 80, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd11b, by Thermo Fisher Scientific (1 mentions)
Facscan, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Fitc conjugated anti cd4, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd8, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Monensin, by Merck Group (1 mentions)
Phorbol 2 myristate 13 acetate, by Merck Group (1 mentions)
7 protocols using pe conjugated anti cd3
1
Hematopoietic Stem Cell Transplantation
2
Characterization of Activated Cytotoxic T Cells
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CTLs were stained with CD3, CD8 and CD69 anti-human mouse antibodies (mAbs), washed, and analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ). The CD3+ CD8+ cells were gated and evaluated for activation cell subsets (CD69+). Briefly, T cells that were maintained under the same culture conditions but without any peptide stimulation were used as negative controls. CTLs were co-incubated at a 1:1 ratio with stimulator cells in round-bottom 96-well plates. After 1 h of incubation, 10 μg/mL Brefeldin A (eBioscience, San Diego, USA) was added to each well. After 5 h of additional incubation, the cells were centrifuged, washed, and stained with phycoerythrin (PE)-conjugated anti-CD3 (eBioscience) and peridinin-chlorophyll protein (PerCP)-conjugated anti-CD8 anti-human mAbs (Biolegend) for 30 min on ice in the dark. After surface staining, cells were washed, fixed, permeabilized, washed with Perm/Wash solution (BD Biosciences), stained with an anti-IFN-γ mAb (eBioscience), and analyzed by flow cytometry38 (link).
3
Multiparametric Flow Cytometry Immunophenotyping
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Single cell suspensions (0.5 x 106 cells) were incubated for 15 min with Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences Pharmingen, San Diego, CA, USA), washed with PBS supplemented with 2% FCS and then stained for 30 min with the indicated conjugated antibodies. Cells were washed twice and fixed with 0.37% formaldehyde in PBS. FITC-conjugated anti-CD19, PE-conjugated anti-CD3, APC-conjugated anti-CD4, BV410-conjugated anti-CD8, APC-conjugated anti-CD11b, BV711-conjugated anti-CD11c, FITC-conjugated anti-Gr-1 and PE-conjugated anti-F4/80, were purchased from eBioscience (San Diego, CA, USA). Cells were analysed in a FACS Fortessa flow cytometer and data were processed with the FlowJo software (Becton Dickinson Labware, Franklin Lakes, NJ, USA).
4
Flow Cytometric Analysis of Lymphocyte Populations
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Antibodies used for flow cytometry included phycoerythrin (PE)-conjugated anti-CD3, FITC-conjugated anti-CD4, FITC-conjugated anti-CD8, or PE-Cy5-conjugated anti-B220 and anti-rat Foxp3 (eBioscience, San Diego, CA, USA). Splenocytes and cells isolated from lymph nodes of the kidney were incubated at 4°C for 20 min with FITC-or PE-conjugated antibody to CD3 or B220 (CD45RA) in the presence of Fc block. In order to identify lymphocyte populations, we first gated cells based on forward and side scatter properties. Next, we identified specific cell populations among these lymphocyte populations using fluorescence-labeled mAbs. The percentages of CD3+B220+, CD3+Foxp3+, and CD3+CD4−CD8− cells in CD3+ cells were determined. The expression was analyzed on a FACScan (Becton Dickinson, Tokyo, Japan) using the CellQuest software (version 3.3; Becton Dickinson, San Jose, CA, USA). Lymphoproliferation in SCG/Kj mice is characterized by the accumulation of Thy-1+, Ly-1+, CD4/CD8 double negative, and B220+ T cells.
5
Analyzing Immune Cell Subsets in MLN
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Single-cell suspensions from the MLN were prepared and enriched by nylon wool. Referring to previous studies [25 (link),26 ], we calculated the frequency of CD4+ CD25+FOXP3+ Treg cells in individual samples, as well as that of the effector T cells Th1 (CD3+CD8–interferon [IFN]-γ+), Th2 (CD3+CD8–IL-4+), Th9 (CD3+CD8–IL-9+), Th17 (CD3+CD8–IL-17A+), and Th22 (CD3+CD8–IL-22+IL-17A–). Cells were stained in PBS containing 1% fetal calf serum with the following antibodies (all from eBioscience): phycoerythrin (PE)-conjugated anti-CD4, fluorescein isothiocyanate (FITC)-conjugated anti-CD25, and PE-Cy5–conjugated anti-FOXP3; PE-conjugated anti-CD3, allophycocyanin-conjugated anti-CD8, FITC-conjugated anti–IFN-γ, anti–IL-4, anti–IL-9, anti–IL-17A, and PerCP-eFluor–conjugated anti–IL-22. Homotype-independent antibody was used as the negative control. IFN-γ, IL-4, IL-9, IL-17A, and IL-22 intracellular staining was performed after 4-h stimulation with 25 ng/mL phorbol 2-myristate 13-acetate, 1 mg/mL ionomycin, and 2 nmol/mL monensin (all from Sigma-Aldrich).
6
Isolation of Murine NK Cells
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NK cells were aseptically isolated by mechanical dispersion of the whole GD8 BALB/c spleen in RPMI-1640 medium supplemented with 1% FBS and penicillin/streptomycin (100 U/ml). Cell suspensions were subsequently passed through a 37 μm nylon mesh followed by density gradient separation using HISTOPAQUE 1083 (Sigma-Aldrich), according to the manufacturer's instructions. Briefly, cell suspensions were carefully loaded onto the HISTOPAQUE 1083 surface and centrifuged at 2000 r.p.m. for exactly 30 min at room temperature. After centrifugation, the opaque interface containing the mononuclear cells was carefully aspirated, washed with RPMI-1640 medium and resuspended in PBS containing 0.2% BSA. Cells were then pretreated with anti-mouse CD16/CD32 mAb (eBioscience) for 10 min on ice and incubated with APC-conjugated anti-CD49b (BD Biosciences, San Jose, CA, USA) and PE-conjugated anti-CD3 (eBioscience) mAbs for 30 min at 4 °C. After incubation, cells were washed once with PBS containing 0.2% BSA and sorted by a FACSAria instrument (BD Biosciences, Franklin Lakes, NJ, USA). Postsort NK cell purity was routinely more than 95% (data not shown).
7
CFSE-based T Cell Proliferation Assay
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Single-cell suspensions from spleen of BALB/c mice were obtained by grinding and filtration through nylon mesh. The lymphocytes were enriched with Ficoll (TBDscience). Lymphocytes (1×10 6 /mL) were stained with CFSE and then co-cultured with C57BL/6 bone marrow-derived DCs (1×10 5 /mL) after Mitomycin C treatment (Roche). Five days later, harvested cells were stained with PE-conjugated anti-CD3 (eBioscience) and T cells proliferation was evaluated by flow cytometry.
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