Percp cy5.5 cd3

Manufactured by Thermo Fisher Scientific

PerCP-Cy5.5 CD3 is a fluorescent-conjugated antibody used for the detection and analysis of CD3-positive cells in flow cytometry applications. It combines the properties of PerCP (Peridinin Chlorophyll Protein Complex) and Cy5.5 fluorescent dyes to enable multicolor flow cytometry.

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4 protocols using percp cy5.5 cd3

Flow Cytometry Analysis of Lymphocytes

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Freshly isolated lymphocytes and splenocytes were labeled with PerCP-Cy5.5 CD4 (eBioscience, clone RM4–5), PE Vβ13 (MR12–4, Biolegend), PE-Cy7 CD45.1 (A20, eBioscience), BV650 CD8 (53–6.7, BD Biosciences), PE-Cy7 CD11c (HL3, BD Biosciences), PE CD28 (37.51, eBioscience), PE H-2K^d (SF1–1.1, BD Biosciences), PerCP-Cy5.5 CD3 (eBioscience, 45-0031-82), PE-Cy7 CD25 (eBioscience, 552880), PE FoxP3 (eBioscience, 12-5773-80B), APC NK1.1 (eBioscience, 17-5941-82), PE PD-1 (eBioscience, 12-9985-81), Alexa 647 CD85k, for ILT3 staining (H1.1, Biolegend), UV455 viability dye (eBioscience, ref. 65-0868-14), eFluor 506 viability dye (eBioscience, 65-0866-14), eFluor 670 (eBioscience, 65-0840-85).
Approx. 5×10⁵ cells were labeled for 20 min at 4^oC with the indicated Abs.
Data was acquired on a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software version 9.9.9 (TreeStar, Ashland, OR).
Gating strategies. For proliferation study: Lymphocytes were gated from SSC-A/FSC-A, live cells from SSC-A/UV455, single cells from FSC-H/FSC-A, CD45.1⁺ from SSCA/CD45.1, proliferation of donor specific T-cells from upper right quadrant of CD4⁺Vβ13⁺. For cell populations: Lymphocytes were gated from SSC-A/FSC-A, live dead cells from SSC-A/ UV455, single cells from FSC-H/FSC-A, then cells of interest as described in the Results section.

Plenter R.J., Grazia T.J., Coulombe M.G., Nelsen M.K., Lin C.M., Beard K.S., Kupfer T.M., Zamora M.R., Gill R.G, & Pietra B.A. (2018). Anti-LFA-1 Induces CD8 T-cell Dependent Allograft Tolerance and Augments Suppressor Phenotype CD8 cells. Cellular immunology, 332, 101-110.

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Isolation and Characterization of Adipose-Derived Immune Cells

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The SVF was isolated from whole adipose tissue, as previously described (60 (link)). Briefly, adipose tissue depots were dissected and weighed. Tissue was then mechanically disrupted by mincing, and it was chemically digested by rocking tissue in 1 mg/mL collagenase IV (Sigma-Aldrich) at 37°C for 30 minutes. Cells were then quenched with RPMI + BSA media and filtered through 100 nm mesh prior to RBC lysis and subsequent filtering with 70 nm mesh filters.
Cells were incubated in Fc Block for 5 minutes on ice before staining with indicated antibodies for 30 minutes at 4°C. Anti-mouse antibodies used included the following: AF488-CD4 (catalog 100423), APCcy7-CD8 (catalog 100713), Brilliant Violet 605-CD279 (PD1) (catalog 135219), PE/Cy7-CD28 (catalog 102125), and APC-TCR-b (catalog 109211) from BioLegend, as well as PerCPcy5.5-CD3 (catalog 45-0031-82), APC-CD25 (catalog 17-0251-82), PE-FoxP3 (catalog 12-4771-82), and PEcy7-Ki67 (catalog 25-5698-82) from eBioscience and Live/dead Fixable Dead Cell Violet Stain Kit (catalog L34955) from Invitrogen.
Stained cells were washed twice with FACS buffer and fixed for intracellular staining using a FoxP3 transcription kit (BD Biosciences). Analysis was performed on an LSR Fortessa Flow Cytometer and analyzed with Flow Jo software (Tree Star Inc.).

Porsche C.E., Delproposto J.B., Geletka L., O’Rourke R, & Lumeng C.N. (2021). Obesity results in adipose tissue T cell exhaustion. JCI Insight, 6(8), e139793.

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Flow Cytometry Analysis of B16f10 Tumors

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In this study, all flow cytometry antibodies and agents were purchased from eBiosciences. For mouse samples, B16f10-xenograft tumors were harvested after experiments and then subjected to rapid and gentle stripping, physical grinding and filter filtration to obtain single cell suspension. After getting rid of dead cells with eBioscience Fixable Viability Dye eFluor 780 (65-0865-14) staining, cells were stained with FITC-CD45 (11-0451-81), PerCP/Cy5.5-CD3 (45-0031-80), PE/Cy7-CD8 (25-0081-81) for 30 min. After fixation and permeabilization, intracellular GZMB was stained using APC-GZMB (17-8898-80). Stained samples were analyzed by FACS, and FlowJo software was used for further data analysis.

Chen X., Wang K., Jiang S., Sun H., Che X., Zhang M., He J., Wen Y., Liao M., Li X., Zhou X., Song J., Ren X., Yi W., Yang J., Chen X., Yin M, & Cheng Y. (2022). eEF2K promotes PD-L1 stabilization through inactivating GSK3β in melanoma. Journal for Immunotherapy of Cancer, 10(3), e004026.

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Isolation and Characterization of Immune Cells

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Spleens and LNs (axillary, inguinal and mesenteric LNs) were pressed through nylon nets to prepare single-cell suspensions. Cells were washed, counted after lysing the red blood cells and then stained with PerCP-Cy5.5-CD3, FITC-CD4 or APC-CD25 mAb (eBioscience). CD4⁺CD25^- T cells and CD4⁺CD25⁺ Tregs were respectively isolated by using a FACSAria cell sorter (BD Biosciences, San Jose, CA). Procedure typically yields 99% enriched cells in all cases as determined by FCM analysis.
Antigen presenting cells (APCs) were prepared from spleen cells by negative selection using CD90.2 magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) to deplete T cells as described previously [26 (link),27 (link)], and were then irradiated with 30 Gy at 2.7 Gy/min using a ¹³⁷Cs source (Gammacell 1000 Elite; Nordion International, Kanata, ON, Canada).
To isolate CD11b⁺ Mφ and CD11c⁺ DC, mice splenic mononuclear cells were isolated by using Percoll density-gradient centrifugation as described previously [28 (link)], then incubated with anti-CD11b (Miltenyi Biotec) or anti-CD11c microbeads (Miltenyi Biotec) and captured on MS columns (Miltenyi Biotec) according to the manufacturer’s instructions as previously described [29 (link)]. The purity of the isolated CD11b⁺ Mφ or CD11c⁺ DC was greater than 95%.

Zhou S., Jin X., Chen X., Zhu J., Xu Z., Wang X., Liu F., Hu W., Zhou L, & Su C. (2015). Heat Shock Protein 60 in Eggs Specifically Induces Tregs and Reduces Liver Immunopathology in Mice with Schistosomiasis Japonica. PLoS ONE, 10(9), e0139133.

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