Apccy7 cd8

Manufactured by BioLegend

Sourced in United States

APCcy7-CD8 is a fluorescently-labeled antibody that binds to the CD8 surface antigen. It can be used to identify and quantify CD8+ T cells in flow cytometry applications.

Automatically generated - may contain errors

Lab products found in correlation

7 protocols using apccy7 cd8

Isolation and Purification of PBMC Subsets

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs were obtained by Ficoll–Hypaque density gradient centrifugation. If indicated, CD8⁺ or CD4⁺ T cells were further purified from isolated PBMCs by negative selection with magnetic beads using a CD8⁺ or CD4⁺ T cell Enrichment Kit (Cell purity >95%, Stem Cell Technologies, Vancouver, Canada). The following antibodies were used for immunostaining to isolate cell subtypes: FITC-CD3, APC-cy7-CD8, APC-CD4, PE-cy7-CD14 and 7-AAD (Biolegend, San Diego, CA, USA). CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), and monocytes (CD3⁻CD14⁺) were selected from 7-AAD-negative live PBMCs using a FACSAria™ flow cytometer (BD Biosciences, Franklin Lake, NJ, USA).

Yin L.B., Song C.B., Zheng J.F., Fu Y.J., Qian S., Jiang Y.J., Xu J.J., Ding H.B., Shang H, & Zhang Z.N. (2019). Elevated Expression of miR-19b Enhances CD8+ T Cell Function by Targeting PTEN in HIV Infected Long Term Non-progressors With Sustained Viral Suppression. Frontiers in Immunology, 9, 3140.

+ Open protocol

+ Expand

Isolation and Phenotyping of Human Visceral Adipose Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols

Visceral adipose tissue was collected from male bariatric surgery patients with IRB approval (HUM00074075) from the University of Michigan and Ann Arbor Veterans Affairs Healthcare System. We did not include or exclude any bariatric surgery samples based on race. Sex and racial classifications were made by the participant and extracted from the medical record.
Tissue was finely minced using surgical scissors (DR Instruments) and then digested in 3 mg/mL collagenase II (Invitrogen; 17101015) for 30 minutes. Digested tissue was then processed in the same manner as digested murine adipose tissue to obtain single-cell suspensions of SVF.
Cells were incubated in Fc Block for 5 minutes on ice before staining with indicated antibodies for 30 minutes at 4°C. Anti-human antibodies used included the following: AF488-CD4 (catalog 317419), PerCPy5.5-CD3 (catalog 300429), APC-CD25 (catalog 356109), APCcy7-CD8 (catalog 344713), APC-PD1 (catalog 329907), and PE-FoxP3 (catalog 320107) from BioLegend, and Live/Dead Fixable Dead Cell Violet Stain Kit (catalog L34955) from Invitrogen.

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.

+ Open protocol

+ Expand

PD1 Blockade Restores Adipose T Cell Function

Check if the same lab product or an alternative is used in the 5 most similar protocols

At 6 weeks of age, C57BL/6J mice were fed ND or HFD for 18 weeks to induce ATT impairment. At 24 weeks of age, mice were given injections of anti–mouse PD1 (catalog CD279, clone RMP1-14, Bio X Cell) or IgG2a isotype control, anti-trinitrophenol (clone 2A3, Bio X Cell) at 10 mg/kg every third day for 4 total injections. The day after the last injection, systemic metabolism was assessed by GTT. After refeeding and resting overnight, mice were sacrificed, and a fraction of fresh SVF was stained for flow cytometry analysis to assess basal T cell differences and ensure the PD1 antibody entered adipose tissue and blocked PD1. The following antibodies were used: AF488-CD4 (catalog 100423) and APCcy7-CD8 (catalog 100713) from BioLegend and PerCPcy5.5-CD3 (catalog 45-0031-82), PE-FoxP3 (catalog 12-4771-82), and Alex Fluor 647 anti–rat IgG2a (catalog 407511) from eBioscience. The remaining SVF was used for an ATT activation assay to assess whether PD1 blockade restored ATT inflammatory potential.

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.

+ Open protocol

+ Expand

Isolation and Characterization of Adipose-Derived Immune Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

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.

+ Open protocol

+ Expand

Lificiguat and Immune Cell Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Lificiguat (YC-1) was obtained from Selleck (United States) (Cat: S7958). CoCl₂ · 6H₂O was purchased from Merck (Germany) (Cat: C8661). The following fluorescence-labeled antibodies were purchased from BD (United States), R&D (United States), or Biolegend (United States): Percp-cy5.5-CD4 (GK1.5), APC-PD-1 (29F.1A12), FITC-CD3 (145-2C11), Percp-cy5.5-CD19 (6D6), APC-cy7-CD8 (54-6.7), PE-cy7-CD11b(M1/70), APC-CD62L (MEL-14), PE-CD25(BC96), PE-CD40L (MR1), APC-CD69 (H12F3), APC-IL-17(TC11-18H10.1), BV421-CXCR5 (L138D7), APC-IFN-γ (XMG1.2), PE-IL10 (JES5-16E3), PE-IL4 (11B11), PE-IL2 (JES6-5H4), anti-mouse CD16/CD32 (2.4G2), Alexa Fluor 488-NFATc1(7A6), APC- HIF-1α(241812). Anti-NFATc1 rabbit mAb (D15F1), anti-tubulin antibody (Cat: 2144S), anti-histone H3 rabbit mAb (D1H2), and HRP-labled secondary antibodies (Cat: L3012-2) were purchased from cell signaling technology (CST, United States). The eFluor506-FVD (Cat: 65-0863-14) was purchased from eBioscience (United States).

Wei H., Xie A., Li J., Fang C., Liu L., Xing J., Shi F., Mo F., Chen D., Xie H., Yang Q., Pan X., Tang X, & Huang J. (2022). PD-1+ CD4 T cell immune response is mediated by HIF-1α/NFATc1 pathway after P. yoelii infection. Frontiers in Immunology, 13, 942862.

+ Open protocol

+ Expand

Flow Cytometry and Immunohistochemistry of Tumor Samples

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, United States). For flow cytometry analysis, BV421 CD45, BV605 CD3, BV710 CD3, PE-Cy7 CD4, AF700 CD8, APC-Cy7 CD8, APC CD335 (NKp46), PerCP-Cy5.5 CD62L, BV510 CD44, FITC Ki-67, PE Granzyme B (GrB), BV650 CD11b, PE-Cy7 F4/80, APC GR1, Zombie NIR Fixable Viability Kit, and TruStain FcX™ PLUS (CD16/32) antibodies were purchased from Biolegend (San Diego, CA, United States). For immunohistochemistry, anti-CD3 and anti-CD8α antibodies were purchased from Abcam (Cambridge, MA, United States). Neutralized antibody anti-mouse PD-L1 (clone 10F.9G2), IgG2b isotype control, and anti-mouse CD8α (Clone 53-6.7) were obtained from BioXCell (West Lebanon, NH, United States). The tumor dissociation kit was obtained from Miltenyi Biotec (Bergisch Gladbach, Germany).

Chun J., Park S.M., Yi J.M., Ha I.J., Kang H.N, & Jeong M.K. (2022). Bojungikki-Tang Improves Response to PD-L1 Immunotherapy by Regulating the Tumor Microenvironment in MC38 Tumor-Bearing Mice. Frontiers in Pharmacology, 13, 901563.

+ Open protocol

+ Expand

Flow Cytometric Analysis of Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For flow cytometric analysis, the following monoclonal antibodies (mAbs) and reagents were used: BV650-CD4 and PE-Cy7-CD3 (BD Biosciences, Tokyo, Japan); PE/Cy7-CD4, BV510-CD4, APC-CD8, APC/Cy7-CD8, Alexa700-CD8, FITC-CD11b, PerCp/Cy5.5-CD21, PE-CD23, PE-CD45.1, PE-CD45.2, FITC-B220, APC-B220, Pacific Blue-Thy1.2, Pacific Blue-c-Kit, APC-IL7Rα, PE-EpCAM, FITC-Gr-1, PE/Cy7-Sca-1, PE-Hamster IgG, PE-Notch1, PE-Notch2, PE-Notch3, and PE-Notch4 (BioLegend, Tokyo, Japan); and PerCp/Cy5.5-CD4, PerCp/Cy5.5-CD19, PE-Thy1.2, APC-Thy1.2, APC-PDGFRα, and FITC-TER119 (Thermo Fisher Scientific, Tokyo, Japan). PE/Cy7-CD19 and APC/Cy7-CD45.1 (Thermo Fisher Scientific). Stained cells were measured using FACSVerse (BD Biosciences) or FACSFortessa (BD Biosciences). Data were analyzed using FlowJo software (BD Biosciences).

Hirano K.I., Hosokawa H., Yahata T., Ando K., Tanaka M., Imai J., Yazawa M., Ohtsuka M., Negishi N., Habu S., Sato T, & Hozumi K. (2022). Dll1 Can Function as a Ligand of Notch1 and Notch2 in the Thymic Epithelium. Frontiers in Immunology, 13, 852427.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!