Perforin apc

Manufactured by Thermo Fisher Scientific

Sourced in United States

Perforin-APC is a fluorochrome-conjugated antibody used for the detection and quantification of perforin, a cytotoxic protein involved in the immune response. It provides a reliable method for the analysis of perforin expression in various cell types by flow cytometry.

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6 protocols using perforin apc

Cytokine Profile in Peripheral Blood

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At the beginning and conclusion of the study, peripheral blood samples were drawn into serum separator tubes (SST). LDL-C levels were quantified using the Cobas 6000 analyzer (Roche Diagnostics, Basel, Switzerland) through enzymatic colorimetric techniques.
For the analysis of PBMCs, we followed procedures based on a previously published article by our group (32 (link)). The study utilized several monoclonal antibodies, including anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD57-FITC, anti-interferon (IFN)-γ-PE-Cy7, and anti-tumor necrosis factor (TNF)-α-APC, IL-17A-APC, Granzyme B-PE, perforin-APC, all sourced from eBioscience, San Diego, CA, USA. Following permeabilization, cells were stained for intracellular cytokines and cytotoxic molecules using anti-IFN-γ-PE-Cy7 and anti-TNF-α-APC. Multicolor flow cytometry was performed with the BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was conducted using FlowJo V10 software (FlowJo, LLC, Ashland, OR, USA). Blood samples with a PBMC viability of 80% or more were used for FACS analysis, with 77 patients meeting this criterion at both baseline and follow-up visits.

Ju S.H., Lim J.Y., Song M., Kim J.M., Kang Y.E., Yi H.S., Joung K.H., Lee J.H., Kim H.J, & Ku B.J. (2024). Distinct effects of rosuvastatin and rosuvastatin/ezetimibe on senescence markers of CD8+ T cells in patients with type 2 diabetes mellitus: a randomized controlled trial. Frontiers in Endocrinology, 15, 1336357.

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Multiparametric Flow Cytometry Analysis

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PBMCs were incubated with 20 ng/mL phorbol myristate acetate plus 1 μg/mL ionomycin (MultiSciences, China) for 1 h, and then 2 µmol/L monensin was added for the final 4 h. Cells were stained with fluorochrome-conjugated antibodies to CD3-PerCP/Cy5.5, CD56-FITC or CD8-FITC (Biolegend, San Diego, CA, United States) at 4 ˚C in the dark. Afterwards, cells were fixed and permeabilized followed by intracellular staining for IFN-γ-PE, TNF-α-PE, IL-2-APC, perforin-APC and granzyme B-PE and isotype matched controls (IL-2-APC from eBioscience, United States; others from Biolegend, CA, United States) at 4 ˚C in the dark. Finally, cells were detected using a Canto II flow cytometer.

Wang W.T., Zhao X.Q., Li G.P., Chen Y.Z., Wang L., Han M.F., Li W.N., Chen T., Chen G., Xu D., Ning Q, & Zhao X.P. (2019). Immune response pattern varies with the natural history of chronic hepatitis B. World Journal of Gastroenterology, 25(16), 1950-1963.

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CTL Cytokine and Cytotoxicity Assay

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CTLs were cultured and pretreated with protein transport inhibitor (PTI) and DMSO for 1 h at 37°C and 5% CO₂ before incubating with CFSE‐labeled EG7‐L1 cells for 6 h. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with saponin (Sigma, 47036) and stained with IL‐2‐PE (1:100, eBioscience, 12‐7021‐82), IFNγ‐PE‐Cy7 (1:100, eBioscience, 25‐7311‐82), perforin–APC (1:100, eBioscience, 17‐9392‐80), or GZMB‐Alexa Fluro (1:100, BioLegend, 515405). Cytokine production was measured by flow cytometric analysis gating on CFSE negative population.

Fan Z., Tian Y., Chen Z., Liu L., Zhou Q., He J., Coleman J., Dong C., Li N., Huang J., Xu C., Zhang Z., Gao S., Zhou P., Ding K, & Chen L. (2020). Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors. EMBO Molecular Medicine, 12(6), e11571.

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Comprehensive Immune Profiling of TILs

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Flow cytometry analysis was performed by using a BD FACSVerse (BD Biosciences). Human TILs were stained with the following monoclonal antibodies: CD45-APC-H7, CD3ε-PerCP, CD8α-V500, CD56-PE-Cy7, CD19-FITC, IFN-γ-FITC, IL-17A-V450 (all from BD Pharmingen, Milan, Italy), IL-21-PE, T-bet-PE-Cy7 (clone 4B10), RORγt-APC (clone AFKJS-9), Foxp3-FITC (clone PCH101), IL-22-APC, IL-6 PerCP (both from eBioscience), CD68-APC (Biolegend, San Diego, CA). Mouse LPMCs and TILs were stained with the following monoclonal antibodies: CD45-APC-Cy7, CD4-PerCP, CD8α-FITC, CD49b-PE (clone DX5), FoxP3-APC, IL-17A-PE, IFN-γ-PE-Cy7 (both from BD Pharmingen), perforin-APC, granzyme B-PE-Cy7 (all from eBioscience). In parallel, cells were stained with the respective control isotype antibodies.

De Simone V., Ronchetti G., Franzè E., Colantoni A., Ortenzi A., Fantini M.C., Rizzo A., Sica G.S., Sileri P., Rossi P., MacDonald T.T., Pallone F., Monteleone G, & Stolfi C. (2015). Interleukin-21 sustains inflammatory signals that contribute to sporadic colon tumorigenesis. Oncotarget, 6(12), 9908-9923.

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Multiparametric Flow Cytometry of Tregs

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For analysis of in vivo experiments, cells were stained with antibodies against human CD45 APC (Invitrogen), CD4 ECD (Beckman Coulter), CD3 Pacific Blue (eBioscience) CD8 PE and CD25 PE-Cy7 (BD) and the viability dye 7-AAD (eBioscience). To analyse expression of Treg-associated markers, freshly sorted or expanded Tregs were stimulated for 15h with anti-CD3/anti-CD28 beads at a ratio of 1 bead to 5 cells, or left untreated. Cells were stained with 7-AAD and antibodies against GITR FITC (R&D Systems), CTLA-4 PE, CD69 APC-Cy7, CD25 PE-Cy7 (BD), TIGIT PE, OX-40 FITC, TIM-3 APC, CD39 PE, FOXP3 eFluor 450, Perforin APC (eBioscience) Helios AlexaFluor 647 (Biolegend), and CD4 ECD (Beckman Coulter). For intracellular antigens (FOXP3, Helios, Perforin, CTLA-4), cells were fixed and permeabilized using a Foxp3 Staining Buffer Set (eBioscience). Samples were acquired using a BD FACSCanto (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences). For staining for BCL-XL and MCL1, Abcam anti-BCL-XL FITC (7B2.5; ab26148) and anti-MCL-1 Alexa Fluor 488 (Y37, ab197529) antibodies were used, respectively, following manufacturer's instructions. Briefly, the cells were fixed with 4% paraformaldehyde and permeablized with PBS/0.1% Tween. The cells were then blocked with 10% normal goat serum/0.3 M glycine, followed by incubation with the antibody.

Issa F., Milward K., Goto R., Betts G., Wood K.J, & Hester J. (2019). Transiently Activated Human Regulatory T Cells Upregulate BCL-XL Expression and Acquire a Functional Advantage in vivo. Frontiers in Immunology, 10, 889.

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Multiparameter Immunophenotyping of Immune Cells

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BM and PB samples were incubated for 30 minutes at room temperature with the following specific antibodies: CD3-Peridinin-chlorophyll-protein (PerCP); CD4-Fluorescein isothiocyanate (FITC) or tandem fluorochrome composed of R-phycoerythrin (PeCy5); CD8-FITC or R-phycoerythrin (PE); CD14-FITC; CD16-PE or Alexa^®647; CD19-PeCy5; CD25-FITC or Allophycocyanin (APC); CD27-PE; CD34-FITC; CD38-PE; CD45-PE, PerCP or APC; CD45RA-APC; CD45RO-APC; CD56-FITC; CD184 (CXCR4)-APC. After incubation, red blood cells were lysed using FACS™ lysing solution (BD Biosciences). For intracellular staining, cells were fixed with 4% paraformaldehyde, and then suspended in permeabilization solution (BD Biosciences) for 30 minutes at room temperature before labelling with granzyme B-PE or APC and perforin-APC (e-Bioscience), or an isotype control antibody (BD Biosciences). For forkhead box P3 (FOXP3)-PE staining, cells were incubated with FOX Buffer solution (BD Biosciences) following the manufacturer’s instructions. Unless specified, all antibodies were from BD Biosciences. For ROS measurement, cells were incubated with 25 µmol/l of 2′,7′-dichlorofluorescein diacetate (DCFDA)-FITC at 37°C for 30 minutes. Fluorescence analysis was performed using a FACS Calibur and analyzed with BD FACS Diva or FlowJo software (Becton Dickinson).

Calgarotto A.K., Longhini A.L., Pericole de Souza F.V., Duarte A.S., Ferro K.P., Santos I., Maso V., Olalla Saad S.T, & Torello C.O. (2021). Immunomodulatory Effect of Green Tea Treatment in Combination with Low-dose Chemotherapy in Elderly Acute Myeloid Leukemia Patients with Myelodysplasia-related Changes. Integrative Cancer Therapies, 20, 15347354211002647.

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