Anti mouse cd8, by BioLegend - Product details

1

Multiparametric Flow Cytometry Analysis of Tumor Immune Microenvironment

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Stromal cells were collected by trypsin and prepared by passing cells through a 40-µm cell strainer. The resulting single-cell suspensions were stained in FACS buffer with the following antibodies for flow cytometric analysis: anti-mouse CD3 (BD Bioscience), and anti-mouse CD45, CD31, Podoplanin, LTβR, VCAM-1 (All from BioLegend). TLSs were dissected from mice, mechanically dissociated and digested with tumor digestion buffer and GentleMACS (Miltenyi Biotec). After lysis of RBCs, the single-cell suspensions were analyzed by flow cytometry with the following antibodies: anti-mouse CD3, CD4, CD11b, CD11c (All from BD Bioscience), anti-mouse CD8, CD19, CD45, CD31, Podoplanin (All from BioLegend), and anti-NK1.1 (eBioscience). MC38 tumors were processed as above and stained with the following antibodies: anti-mouse CD3, CD4, CD69, CD27, CD45RA, PD-1, LAG3, and CD127 (All from BD Bioscience), and anti-mouse CD8, CD62L, CD44, KLRG1, CTLA-4, Tim-3, and CD45.2 (All from BioLegend). DAPI (Sigma-Aldrich) was used as a cell viability marker. The cells were analyzed by the LSR II flow cytometry equipped with five lasers (BD Biosciences), and the data were analyzed with Flow Jo (Tree Star).

Zhu G., Nemoto S., Mailloux A.W., Perez-Villarroel P., Nakagawa R., Falahat R., Berglund A.E, & Mulé J.J. (2018). Induction of Tertiary Lymphoid Structures With Antitumor Function by a Lymph Node-Derived Stromal Cell Line. Frontiers in Immunology, 9, 1609.

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Comprehensive Immune Cell Profiling

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The following antibodies were used for the present study.
Anti-mouse CD3, clone number 17A2, BioLegend, 05112-40-100, V450;
Anti-mouse CD4, clone number GK1.5, BioLegend, 100434, PerCP/Cyanine5.5;
Anti-mouse CD8, clone number 53-6.7, BioLegend, 100714, APC/Cyanine7;
Anti-mouse CD25, clone number 3C7, BioLegend, 101908, FITC;
Anti-mouse CD69, clone number H1.2F3, BioLegend, 104508, PE;
Anti-mouse CTLA-4, clone number UC10-489, BioLegend, 106305, PE;
Anti-mouse PD-1, clone number 29F.1A12, BioLegend, 135210, APC;
Anti-mouse CD19, clone number 1D3, eBioscience, 12-0193-82, PE;
Anti-mouse CD45, clone number 30F11, BioLegend, 103122, Alexa Fluor 488;
Anti-human CD45, clone number HI30, BioLegend, 304025, PerCP;
Anti-mouse IFNγ, clone number XMG1.2, eBioscience, PE;
Anti-mouse IL-17A, clone number TC11-18H10.1, BioLegend, Alexa Fluor 647;
Anti-mouse IL-13, clone number 13A, BioLegend, PE/Cyanine7;
Anti-mouse Foxp3, clone number MF14, BioLegend, PE;
Anti-mouse TNFα, clone number MP6-XT22, BioLegend, Alexa Fluor 488;
Purified Anti-mouse IFNγ antibody, clone number XMG1.2;
Purified anti-mouse IL-4 antibody, clone number 11B11.

Yang F.M., Shen L., Fan D.D., Chen K.H, & Lee J. (2022). DMGV Is a Rheostat of T Cell Survival and a Potential Therapeutic for Inflammatory Diseases and Cancers. Frontiers in Immunology, 13, 918241.

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3

CD8+ T cell Adoptive Transfer and Analysis

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Total CD8⁺ T cells were purified from spleens and lymph nodes of 6–8wk old Klrk1^−/− or wild-type NOD mice by negative selection using magnetic beads (BD Biosciences) according to the manufacturer’s protocol. Isolated Klrk1^−/− and wild-type CD8⁺ T cells were then labeled with eFluor 670 (eBioscience) and CFSE (Thermo Fisher Scientific), respectively. The labeled cells were then mixed 1:1 and adoptively transferred via retro orbital injection into 6–8 wk old wild-type NOD recipient mice (Supplemental Fig. 2A). Total splenocytes were harvested after 7 d, incubated for 4–6 h in complete media with GolgiPlug protein transport inhibitor (BD Biosciences), followed by fixation and permeabilization using BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s directions. Cells were then stained overnight at 4°C with anti-mouse TNF-α (BD Pharmingen), anti-mouse IFN-γ (BD Pharmingen), and anti-mouse CD8 (BioLegend), and analyzed by flow cytometry. Transferred CD8⁺ T cells were analyzed by gating on live lymphocytes based on forward and side scatter, CD8⁺ cells, then either eFluor 670⁺ or CFSE⁺ cells.

Trembath A.P., Sharma N., Raju S., Polić B, & Markiewicz M.A. (2017). A Protective Role for NKG2D–H60a Interaction via Homotypic T Cell Contact in Nonobese Diabetic Autoimmune Diabetes Pathogenesis. ImmunoHorizons, 1(9), 198-212.

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4

Quantification of Effector Memory CD8+ T Cells

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Surface stained Ova-specific effector memory CD8⁺ T cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences), followed by staining with rabbit anti-mouse HIF1α antibody (Novus Biologicals#nb100-449) or mouse anti-mouse TFAM (Santa Cruz Biotechnology#sc-166965) at 4°C for 1 hour. This was followed by staining with Alexa Fluor conjugated secondary antibodies (Molecular Probes) at 4°C for 1 hour. In case of intracellular staining of IL-2 and IFN-γ, splenocytes harvested from CD45.1⁺ mice were pre-treated with FcRγII/III (Fc blocker) and IgG_2b anti-mouse CD16/CD32 antibodies, then stained with anti-mouse CD8 (Biolegend#100725), anti-mouse CD45.2 (BD PharMingen#561096) and anti-mouse Ova_tetramer before intracellular staining. Surface stained splenocytes were then fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences#554714), followed by staining with anti-mouse IL-2 (BD PharMingen#554428) or anti-mouse IFN-γ (Biolegend# 505806). In case of intranuclear staining for TCF7, surface stained cells were fixed and permeabilized using Foxp3/Transcription factor staining buffer set (eBioscience#00-5523-00), followed by staining with anti-mouse TCF7/TCF1 antibody (R&D systems#FAB8224R). Stained cells were analyzed on BD FACSCantoII or BD LSRII.

Gupta S.S., Sharp R., Hofferek C., Kuai L., Dorn GW I.I., Wang J, & Chen M. (2019). NIX-Mediated Mitophagy Promotes Effector Memory Formation in Antigen-Specific CD8+ T Cells. Cell reports, 29(7), 1862-1877.e7.

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5

Quantification of Antigen-Specific CD8+ T Cell Cytotoxicity

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P14 CD8+ T cells were isolated and activated by pMHC liposomes or anti-CD3/anti-CD28 as described above. The media was changed and supplemented with 30 U/ml rhIL-2 after 48 hr. On day 5 post-activation, MC38 target cells were pulsed with GP33 peptide in OptiPRO SFM media (Gibco) for 1 hr at 37°C. Pulsed MC38 cells were washed 5 times with PBS, seeded in a 96 well plate at 6×10⁵ cells/ml in T cell media, and incubated for 4 hr to allow adhesion. Activated P14 T cells were washed 5 times with PBS and incubated with MC38 cells at a 5:1 effector-to-target ratio overnight. For analysis of IFNγ expression, cells were washed with PBS, blocked with Fc Shield (Tonbo) for 10 minutes on ice, and stained with anti-mouse CD8 (Biolegend, Clone: 53–6.7) and anti-mouse IFNγ (Biolegend, Clone: XMG1.2) for 30 min on ice before collection on a BD Accuri C6.

Dahotre S.N., Romanov A.M., Su F.Y, & Kwong G.A. (2021). Synthetic Antigen-Presenting Cells for Adoptive T Cell Therapy. Advanced therapeutics, 4(8), 2100034.

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6

Tumor-Infiltrating Lymphocyte Analysis Protocol

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Tumor-infiltrating lymphocytes were analyzed as previously described^{36 (link),37 (link)}. Briefly, 4 days after the last injection, the tumor masses were harvested from euthanized mice and weighed. Then, the tumor tissues were minced and passed through a 70-μm pore filter. The separated single cells were stained with the following antibodies: anti-mouse CD3, anti-mouse CD4, anti-mouse CD8, anti-mouse Foxp3, anti-mouse IFN-γ and anti-mouse TNF-α (BioLegend). Intracellular staining of tumor-infiltrating lymphocytes was conducted according to the manufacturer’s protocol. The staining results were analyzed using FlowJo software (TreeStar Inc.).

Hong J., Jung M., Kim C., Kang M., Go S., Sohn H., Moon S., Kwon S., Song S.Y, & Kim B.S. (2023). Senescent cancer cell-derived nanovesicle as a personalized therapeutic cancer vaccine. Experimental & Molecular Medicine, 55(3), 541-554.

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7

Tumor Dissociation and Immune Cell Analysis

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Tumors were surgically removed from animals and were place in cold PBS. Following, tumors were mechanically shredded using scalpel and incubated in RPMI medium containing 2mg/ml Collagenase D (Roche), and 2 mg/ml DNaseI (Roche) in 37°C for 20min. The resulting cell suspension was washed with PBS, passed through a 70-μm filter (Falcon) and cells were incubated in FACS buffer (PBS supplemented with 1%BSA (Sigma), 2mM EDTA and 0.05% sodium azide) in the presence of staining antibody. For intracellular staining, the Cytofix/Cytoperm Kit (Fisher Scientific) was used according to the manufacturer’s instruction. Cells were acquired on FACSCanto, LSRII, and LSRFortessa systems (BD) and analyzed with FlowJo software (Tree Star). Antibodies used for flow cytometry were: anti-mouse CD8, CD4, TCRβ, CD107a, Granzyme B, and IFN-γ (all Biolegend).

Wolf Y., Bartok O., Patkar S., Eli G.B., Cohen S., Litchfield K., Levy R., Jiménez-Sánchez A., Trabish S., Lee J.S., Karathia H., Barnea E., Day C.P., Cinnamon E., Stein I., Solomon A., Bitton L., Pérez-Guijarro E., Dubovik T., Shen-Orr S.S., Miller M.L., Merlino G., Levin Y., Pikarsky E., Eisenbach L., Admon A., Swanton C., Ruppin E, & Samuels Y. (2019). UVB-Induced Tumor Heterogeneity Diminishes Immune Response in Melanoma. Cell, 179(1), 219-235.e21.

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8

Multiparametric Flow Cytometry Analysis

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For the analysis of cell surface molecules, single-cell suspensions were prepared and fluorescently stained with the following antibodies from BioLegend: anti-human CD3 (317305), anti-human CD4(357146), anti-human CD8(344712), anti-human CD25(302609), anti-human CD56(362505), anti-mouse CD3(100203), anti-mouse CD4(100413), and anti-mouse CD8(100721). Tregs were first incubated with surface markers, washed, resuspended in 1 ml of cold Fixation/permeabilization buffer (eBioscience), and incubated at 4°C for 1 h. After washing with 2 ml of permeabilization buffer (eBioscience), cells were stained with Foxp3 antibodies at 4°C for 50 min protected from light. Finally, cells were washed with 2 ml of permeabilization buffer and detected on FACS CantoII (BD Biosciences). All flow cytometer data were analyzed by the FlowJo software.
The concentrations of IL1/2/4/6/8/10 and TNF-α in the serum of patients were measured using the Bio-Plex Pro Human Cytokine 8-plex assay according to the manufacturer’s instructions.

Wang J., Liu J., Wang M., Zhao F., Ge M., Liu L., Jiang E., Feng S., Han M., Pei X, & Zheng Y. (2022). Levamisole Suppresses CD4+ T-Cell Proliferation and Antigen-Presenting Cell Activation in Aplastic Anemia by Regulating the JAK/STAT and TLR Signaling Pathways. Frontiers in Immunology, 13, 907808.

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9

CD8+ T cell Adoptive Transfer and Analysis

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Total CD8⁺ T cells were purified from spleens and lymph nodes of 6–8wk old Klrk1^−/− or wild-type NOD mice by negative selection using magnetic beads (BD Biosciences) according to the manufacturer’s protocol. Isolated Klrk1^−/− and wild-type CD8⁺ T cells were then labeled with eFluor 670 (eBioscience) and CFSE (Thermo Fisher Scientific), respectively. The labeled cells were then mixed 1:1 and adoptively transferred via retro orbital injection into 6–8 wk old wild-type NOD recipient mice (Supplemental Fig. 2A). Total splenocytes were harvested after 7 d, incubated for 4–6 h in complete media with GolgiPlug protein transport inhibitor (BD Biosciences), followed by fixation and permeabilization using BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s directions. Cells were then stained overnight at 4°C with anti-mouse TNF-α (BD Pharmingen), anti-mouse IFN-γ (BD Pharmingen), and anti-mouse CD8 (BioLegend), and analyzed by flow cytometry. Transferred CD8⁺ T cells were analyzed by gating on live lymphocytes based on forward and side scatter, CD8⁺ cells, then either eFluor 670⁺ or CFSE⁺ cells.

Trembath A.P., Sharma N., Raju S., Polić B, & Markiewicz M.A. (2017). A Protective Role for NKG2D–H60a Interaction via Homotypic T Cell Contact in Nonobese Diabetic Autoimmune Diabetes Pathogenesis. ImmunoHorizons, 1(9), 198-212.

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10

Quantification of Effector Memory CD8+ T Cells

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Surface stained Ova-specific effector memory CD8⁺ T cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences), followed by staining with rabbit anti-mouse HIF1α antibody (Novus Biologicals#nb100-449) or mouse anti-mouse TFAM (Santa Cruz Biotechnology#sc-166965) at 4°C for 1 hour. This was followed by staining with Alexa Fluor conjugated secondary antibodies (Molecular Probes) at 4°C for 1 hour. In case of intracellular staining of IL-2 and IFN-γ, splenocytes harvested from CD45.1⁺ mice were pre-treated with FcRγII/III (Fc blocker) and IgG_2b anti-mouse CD16/CD32 antibodies, then stained with anti-mouse CD8 (Biolegend#100725), anti-mouse CD45.2 (BD PharMingen#561096) and anti-mouse Ova_tetramer before intracellular staining. Surface stained splenocytes were then fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences#554714), followed by staining with anti-mouse IL-2 (BD PharMingen#554428) or anti-mouse IFN-γ (Biolegend# 505806). In case of intranuclear staining for TCF7, surface stained cells were fixed and permeabilized using Foxp3/Transcription factor staining buffer set (eBioscience#00-5523-00), followed by staining with anti-mouse TCF7/TCF1 antibody (R&D systems#FAB8224R). Stained cells were analyzed on BD FACSCantoII or BD LSRII.

Gupta S.S., Sharp R., Hofferek C., Kuai L., Dorn GW I.I., Wang J, & Chen M. (2019). NIX-Mediated Mitophagy Promotes Effector Memory Formation in Antigen-Specific CD8+ T Cells. Cell reports, 29(7), 1862-1877.e7.

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Anti mouse cd8

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18 protocols using anti mouse cd8

Multiparametric Flow Cytometry Analysis of Tumor Immune Microenvironment

Comprehensive Immune Cell Profiling

CD8+ T cell Adoptive Transfer and Analysis

Quantification of Effector Memory CD8+ T Cells

Quantification of Antigen-Specific CD8+ T Cell Cytotoxicity

Tumor-Infiltrating Lymphocyte Analysis Protocol

Tumor Dissociation and Immune Cell Analysis

Multiparametric Flow Cytometry Analysis

CD8+ T cell Adoptive Transfer and Analysis

Quantification of Effector Memory CD8+ T Cells

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