Cd8 percp efluor 710

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD8-Percp-eFluor 710 is a fluorochrome-conjugated antibody used in flow cytometry applications to identify and quantify CD8-positive cells in a sample. The Percp-eFluor 710 fluorophore is used to label the antibody, providing a specific signal detectable by flow cytometry instruments.

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PerCP-eFluor 710 (33 citations) CD8-PerCP (13 citations)

6 protocols using cd8 percp efluor 710

ELISPOT and Flow Cytometry Analysis of IFN-γ, IL-4, and TNF-α Production

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Spleen lymphocytes were seeded into anti-mouse IFN-γ mAb pre-coated 96-well plates at 2 × 10⁵ cells (100 μL/well) and stimulated with 10 μg/mL purified protein for overnight incubation; phorbol 12-myristate 13-acetate (PMA) was used as a positive control. The ELISPOT assay was performed using a mouse IFN-γ ELISPOT Kit (Dakewe) according to the manufacturer's instructions (Ranieri et al., 2014 ). Spots were counted using an immunospot reader (Cellular Technology Limited, USA).
Spleen lymphocytes (2 × 10⁶ cells) were firstly stimulated for 2 h with 10 μg/mL of purified E1-DIII + NS1-2 and E4-DIII + NS1-3 protein as a specific antigen. Then the treated cells were continue cultured in the presence of 10 mg/mL monensin and brefeldin A in complete RPMI 1640 (Gibco) overnight. Stimulated cells were first incubated and stained with Fixable Dye eFluor 506 and the CD8-Percp-eFluor 710, CD3e-eFluor 450 (eBioscience), CD45-APC-Cy7, and CD4-FITC (BD Biosciences) surface markers. Then, cells were fixed in permeabilization buffer (Thermo Fisher Scientific) and stained with IFN-γ-PE-eFlour 610, IL-4-PE-Cyanine7, and TNF-α-APC (eBiosciences). All labeled lymphocytes were analyzed on a FACSAria III flow cytometer (BD Biosciences), and the data were analyzed using FlowJo V10.

He L., Sun W., Yang L., Liu W, & Li J. (2022). A multiple-target mRNA-LNP vaccine induces protective immunity against experimental multi-serotype DENV in mice. Virologica Sinica, 37(5), 746-757.

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BMDC-mediated OT-I CD8+ T cell activation

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Day 6 BMDCs were dosed
with SIINFEKL or NP samples for 4 h, washed, and further incubated
at 37 °C for 23 h. OT-I CD8+ T cells were isolated from spleens
of OT-I mice using mouse CD8 T cell isolation kit (Miltenyi Biotec)
and labeled with CellTrace Violet Cell Proliferation Kit (Invitrogen)
for 10 min at 37 °C. Labeled OT-I cells were then added to treated
BMDCs and co-incubated for 72 h at 37 °C. After the incubation,
cells were stained with CD8-PerCP-eFluor710, CD3-FITC, and CD11c-PE
(eBioscience) and examined for T cell proliferation with Cyan ADP
followed by analysis with Summit software.

Kapadia C.H., Tian S., Perry J.L., Luft J.C, & DeSimone J.M. (2019). Role of Linker Length and Antigen Density in Nanoparticle Peptide Vaccine. ACS Omega, 4(3), 5547-5555.

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Characterization of Antigen-Specific T Cell Responses

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Antigen-specific CD4⁺ and CD8⁺ T lymphocyte immune responses were characterized by the ICS assay. In brief, mouse spleens were removed under aseptic conditions and stimulated with the RBD of the S protein (10 μg/mL, Sino Biological, China). Then, the cells were incubated with GolgiPlug (BD Biosciences, USA), incubated and stained with Fixable Dye eFluor 506, and the CD8-Percp-eFluor 710, CD3e-eFluor 450 (eBioscience, USA), CD45-APC-Cy7, and CD4-FITC (BD Biosciences, USA) surface markers. The cells were then fixed in permeabilization buffer (Thermo Fisher Scientific, USA) and stained with IFNγ-PE-eFlour 610, IL4-PE-Cyanine7, and TNFα-APC (eBiosciences, USA). All labeled lymphocytes were analyzed on a FACSAria III flow cytometer (BD Biosciences), and the data were analyzed using FlowJo V10.

Sun W., He L., Zhang H., Tian X., Bai Z., Sun L., Yang L., Jia X., Bi Y., Luo T., Cheng G., Fan W., Liu W, & Li J. (2021). The self-assembled nanoparticle-based trimeric RBD mRNA vaccine elicits robust and durable protective immunity against SARS-CoV-2 in mice. Signal Transduction and Targeted Therapy, 6, 340.

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Anti-PD-1 and Anti-CTLA4 Antibody Protocol

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Therapeutic anti-PD-1 (clone RMP1-14) and anti-CTLA4 (9H10) monoclonal antibodies were produced by BioXcell. Antibodies used for flow cytometry were purchased from the following sources (dilutions are indicated in parentheses): eBioscience (CD45.2 Alexa Fluor 700, cat: 56-0454 (1:200), CD3 PE-Cy7, cat: 25-0031 (1:200), CD4 ef450, cat: 48-0041 (1:200), CD8 PerCP-efluor710, cat: 46-0083 (1:200), CD11b APC-efluor 780, cat: 47-0112 (1:600), ICOS PE, cat: 12-5985 (1:200), PD-L1 PE Cy7, cat: 25-5982-82 (1:200), FoxP3 Alexa Fluor 700, cat: 56-5773 (1:100), FoxP3 APC, cat: 17-5773 (1:200), PD-1 PE-Cy7, cat: 25-9985 (1:200)), Invitrogen (Granzyme B PE-Texas Red, cat: GRB17 (1:125)) and BD Pharmingen (Ki67-Alexa Fluor 488, cat: 561165 (1:50)).

Oseledchyk A., Ricca J.M., Gigoux M., Ko B., Redelman-Sidi G., Walther T., Liu C., Iyer G., Merghoub T., Wolchok J.D, & Zamarin D. (2018). Lysis-independent potentiation of immune checkpoint blockade by oncolytic virus. Oncotarget, 9(47), 28702-28716.

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Immunotherapeutic Response Evaluation

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The commercial sources for reagents were as follows: CpG oligodeoxynucleotide ODN2216 (Invitrogen); We used the following antibodies. Therapeutic anti-CTLA4 (clone 9H10 and 9D9), anti-PD1 (clone RMP1-14), anti-PD-L1 (clone 10F.9G2) were purchased from BioXcell; Antibodies used for flow cytometry were purchased from eBioscience (CD45.2 Alexa Fluor 700, CD3 PE-Cy7, CD4 APC-efluor780, CD8 PerCP-efluor710, FOXP3 Alexa Fluor 700, MHC Class I APC, CD40 APC, CD80 APC, CD86 APC), Invitrogen (CD4 QDot 605, Granzyme B PE-Texas Red, Granzyme B APC), BD Pharmingen (Ki-67-Alexa Fluor 488).

Dai P., Wang W., Yang N., Serna-Tamayo C., Ricca J.M., Zamarin D., Shuman S., Merghoub T., Wolchok J.D, & Deng L. (2017). Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic antitumor immunity via STING and Batf3-dependent dendritic cells. Science immunology, 2(11), eaal1713.

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Multiparameter Flow Cytometric Analysis

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The commercial sources for reagents were as follows: Antibodies used for flow cytometry were purchased from eBioscience (Live/Dead eFluor 506, CD45.2 Alexa Fluor 700, CD3 PE-Cy7, CD4 Pacific blue-eFluor 450, CD8 PerCP-efluor710, CD11b APC-eFluor 780, MHC Class I APC, CD40 APC, CD80 APC, CD86 APC), Invitrogen (granzyme B PE-Texas Red), and BD Pharmingen (CD11c-PE-Cy7). Murine anti-GM-CSF antibody was purchased from Thermo Fisher. DNAse I and Liberase TL were purchased from Roche. Recombinant murine GM-CSF protein was purchased from GenScript. Therapeutic anti-CTLA4 (clone 9H10 and 9D9) and anti-PD-L1 (clone 10F.9G2) were purchased from BioXcell.

Wang W., Liu S., Dai P., Yang N., Wang Y., Giese R.A., Merghoub T., Wolchok J, & Deng L. (2021). Elucidating mechanisms of antitumor immunity mediated by live oncolytic vaccinia and heat-inactivated vaccinia. Journal for Immunotherapy of Cancer, 9(9), e002569.

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