CD8+ T cells were isolated from the TGs of VEGF-Aflox/flox mice at day 7 pi using the MACS column-based CD8a T cell isolation kit (Miltenyibiotec). Approximately 5 × 105 isolated CD8+ T cells were then cultured in a 24-well plate in the presence or absence of PMA/Ionomycin (EMD Millipore) or DMSO (Sigma-Aldrich) vehicle as previously described49 . The cells were then harvested and intracellularly stained with anti-interferon-γ APC (eBioscience, Catalogue # 17-7311) followed by surface stain for CD3, CD8, and CD45, antigen expression 3 hr post-stimulation.
Pma ionomycin
Manufactured by Merck Group
Sourced in United States, United Kingdom, Canada, Germany
PMA/ionomycin is a combination of two chemical compounds commonly used as a laboratory tool to stimulate cellular activation and induce the production of specific proteins or cytokines. PMA (Phorbol 12-Myristate 13-Acetate) is a protein kinase C activator, while ionomycin is a calcium ionophore. Together, they provide a potent stimulus for various cell types, including T cells and other immune cells. This product is primarily used in research applications to study cellular signaling pathways and immune function.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (14 mentions)
Golgiplug, by BD (12 mentions)
Golgistop, by BD (9 mentions)
Cytofix cytoperm, by BD (6 mentions)
Fc500 flow cytometer, by Beckman Coulter (4 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Cytofix cytoperm kit, by BD (4 mentions)
Lsrii flow cytometer, by BD (4 mentions)
Cxp software, by Beckman Coulter (3 mentions)
Facscanto, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Facsaria, by BD (3 mentions)
Il 12, by R&D Systems (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Hanks balanced salt solution (hbss), by Wisent (3 mentions)
Brefeldin a, by BD (3 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Anti interferon γ apc, by Thermo Fisher Scientific (2 mentions)
Cd8a t cell isolation kit, by Miltenyi Biotec (2 mentions)
86 protocols using pma ionomycin
1
Interferon-γ Production in CD8+ T Cells
2
Interferon-γ Production in CD8+ T Cells
3
Identification of EBOV T Cell Epitopes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The peptide epitopes of EBOV GP and VP40 proteins that are specific for CD8+ T lymphocytes in BALB/c mouse (GP-T1: LYDRLASTV; GP-T2: GPCAGDFAF; VP40-T1: YFTFDLTALK; VP40-T2: TSPEKIQAIM) were selected according to previous reports (Warfield et al., 2005 (link); Wu et al., 2012 (link)), and synthesized (GeneScript, Nanjing, China). After the preparation of single cell suspensions from mouse splenocytes, ELISPOT assays were performed in pre-coated 96-well plates (Dakewe Biotech, China). The antibody-coated plates were blocked with complete RPMI 1640 medium for 2 h at room temperature. After blocking, 100 μl of splenocytes suspension (1 × 106 cells/ml) containing different peptides (10 μg/ml) were added to each well. A positive control PMA/ionomycin (Sigma) and a media negative control were included in all assays. The plates were incubated for 24 h in a humidified incubator at 37°C, 5% CO2. Plates were then washed and processed according to manufacturer’s instructions, and spots were enumerated using an ImmunoSpot reader and ImmunoSpot software (Cellular Technology Ltd.). Peptide-specific CD8+ T-cell frequency was expressed as Spots forming cells (SFCs)/1 × 105 splenocytes. Background spots (negative control wells) were subtracted from test wells. A positive response to a peptide was defined as having > 5 SFCs/1 × 105 splenocytes after subtraction of the background.
4
Multiparametric flow cytometry of T-cell subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used flow cytometry analysis to assess the production of FOXP3, Helios, GATA3, IL-9, IL-17A, and IL-10 by CXCR6+ and CD4+ T cells. Briefly, splenocytes were incubated with phorbol 12-myristate 13-acetate (PMA)/ionomycin (Sigma-Aldrich) for 4 h in the presence of brefeldin-A (GolgiPlug, BD Biosciences), which prevents the transport of cytokines and transcription factors out of the cell [12 (link),33 (link),41 (link)]. Cells were washed and surface stained for CD4, and CXCR6 surface receptors (BioLegend, San Diego, CA, USA). After permeabilization and fixation (BioLegend), the cells were stained with intracellular cytokines (anti-IL-9, anti-IL-10, and anti-IL-17A; BioLegend) and transcription factors (anti-FOXP3, anti-GATA3, and anti-Helios; BioLegend). The proportions of CXCR6+FOXP3+, CXCR6+Helios+, CD4+GATA3+, CD4+IL-9+, CXCR6+IL-10+, and CD4+IL-17A+ cells were acquired via a FC 500 flow cytometer and analyzed using CXP software (Beckman Coulter, Indianapolis, IN, USA).
5
Analyzing Tumor-Infiltrating Leukocytes by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor tissues were collected from each mouse on Day 8 or 9 after treatment, dissociated into single cells by using a Tumor Dissociation Kit and a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). In cell mixtures, leukocytes positive for CD45 were isolated with mouse TIL (CD45) microbeads (Miltenyi Biotec) by using an OctoMACS Separator (Miltenyi Biotec). After washing and filtration, cells were blocked with Mouse BD Fc Block (BD Biosciences, San Jose, CA, USA), and stained with the antibodies listed in S1 Table and with DAPI (4′,6-diamidino-2-phenylindole, Dojindo, Kumamoto, Japan). The gating strategy is shown in S1 Fig . To detect IFN-γ-producing activated CD8+ T cells, we incubated CD45+ cells with RPMI1640 containing with 10% FBS, PMA/Ionomycin (Sigma, St. Louis, Missouri, USA), and BD GolgiPlug (BD Biosciences) for 4 h at 37 °C, and then stained them with antibodies against CD45, CD3 (Thermo Fisher Scientific, Waltham, MA, USA), CD4, and CD8 (BD Biosciences). The cells were then fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) and then stained with antibody against IFN-γ or GzmB (BD Biosciences). The cells were sorted with a BD FACS AriaII SORP or LSRFortessa X-20 (BD Biosciences) and the data were analyzed by using FlowJo v10 (BD Biosciences) or Cytobank (Cytobank, Inc., Santa Clara, CA, USA).
6
Cytokine Production in Parasite and Allergen Stimulation
7
Isolation and Flow Cytometry of Murine T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions of leukocytes were prepared with complete RPMI inclusive of 5% FCS, 1% penicillin and streptomycin. Pelleted cells were lysed with ACK lysis buffer (Invitrogen), and washed in sterile FACS-buffer comprising PBS with 5% FCS. For T-cell selection, resuspended cells were stained with anti-CD8 APC antibody, and 1 μg propidium iodide (Invitrogen). FACS was used to obtain a >98% pure population of APC+, PI- cells (Moflo, Beckman Coulter, Brea, CA, USA). Cells were washed in sterile PBS in preparation for transfer to recipient mice by tail vein injection, or resuspended in complete RPMI for culture. For flow cytometric examination, surface staining of single-cell suspensions were performed at 4 °C before fixation in 1% formalin in PBS. Where intracellular cytokine staining was required, T cells were pre-stimulated with 25 ng/ml PMA/ionomycin (Sigma-Aldrich) in the presence of 1 μg monensin (Sigma-Aldrich) for 4 h at 37 °C. Cells were then put on ice and surface stained, before incubation in fixation and permeabilisation buffer (BD Systems, Franklin Lakes, NJ, USA) for 30 min before intracellular staining. Flow cytometry was performed using FacsCanto (BD Systems).
8
Stimulation of PBMC with Diverse Antigens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
0.1 × 106 PBMCs in 100 µL of culture medium (2% penicillin/streptomycin, 1% l -glutamine, and 10% FCS in RPMI) were stimulated in 96-well round bottom plates for 16 h with the following antigens: purified protein derivative (PPD) (10 µg/mL, Serum Staten Institute, Denmark), heat-killed Listeria monocytogenes (HKLM) (TLR2 agonist; 109/mL, Invivogen), lipopolysaccharide (LPS) (TLR4 ligand; 1 µg/mL, Invivogen), CLO-75 (TLR7/8 agonist; 10 µg/mL, Invivogen); and heat-killed Streptococcus pneumonia (SP) (105 cells/mL), CA (105 cells/mL), or Escherichia coli (EC) (106 cells/mL). PMA/ionomycin (0.1 μg/1 μg/mL, Sigma, UK) was used as a positive control, and RPMI as a negative control. Supernatants were collected after the addition of 100 µL of RPMI and centrifugation at 1,500 rpm for 10 min. Supernatants were stored at −20°C until needed for cytokine analysis.
9
Characterization of SARS-CoV-2-specific CD4+ T cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The generation of the different SARS-Cov-2-specific CD4+ T cell lines and their specificity and reactivity were confirmed by an immunophenotypic and functional assay where the cell lines were stimulated overnight in 5% CO2 at 37°C with their irradiated autologous feeder cells in the absence (media) or in the presence of their respective antigens (1µg/mL of MP-S or 1µg/mL of MP-M). Both CD4+ TCLs were also stimulated with 1µg/mL MP-CMV and PMA/ionomycin (Sigma-Aldrich) (0.5/0.05 pg/ml). The cells were then stained for viability (Live/Dead fixable blue (UV450), Molecular Probes), and then incubated with anti-CD3 (BV421), anti-CD4 (PerCP-Cy5.5) for 30 min in the dark at room temperature. The cells were next washed twice with FACS buffer, then fixed and permeabilized using a Fix/Perm buffer kit (BioLegend) for 30 min at 4°C. The cells were washed twice with Perm buffer (BioLegend) and resuspended with the intracellular antibody pool containing anti-CD69 (FITC), anti-CD154 (APC), anti-TNF-α (Alexa Fluor 700) and anti-IFN-γ (BUV737) for 30 min at 4°C. Finally, the cells were washed twice with Perm buffer and then acquired using the BD LSRFortessa flow cytometer (BD Biosciences) and FACSDiva software (BD Biosciences) for acquisition. All analyses were performed using FlowJo v10.5.3.
10
Quantifying T Cell Cytokine Profiles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry was used to evaluate IL-17A-, IL-17F-, IL-21-, TNF-α-, STAT3-, and RORγt-expressing CD4+ T cells in spleens. Following the published protocol [10 (link)], splenocytes were incubated with PMA/ionomycin (Sigma-Aldrich, St. Louis, MO, USA), followed by the addition of Golgi-Plug (BD Biosciences, San Jose, CA, USA). Next, cells were rinsed with washing buffer/PBS, collected, and stained with fluorescein isothiocyanate (FITC)-anti-CD4, allophycocyanin (APC)-anti-CD4, PE/Dazzle-anti-CD4, PE/Dazzle-anti-IL-17A, PE-anti-IL-17F, PE-anti-IL-21, PE/Dazzle-anti-TNF-α, PE/Cyanine7-STAT3, and PE-anti-RORγt fluorescent antibodies (BioLegend, San Diego, CA, USA). Cells were analyzed using the FC 500 Flow Cytometer and counted in CXP (Beckman Coulter, Brea, CA, USA).
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!