Splenocytes from immunized and control mice were isolated after the second immunization. Before staining, splenocytes were grown in RPMI 1640 (EuroClone) containing 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies), 100 U/mL penicillin (EuroClone), 100 U/mL streptomycin (EuroClone), 100 μg/mL glutamine (EuroClone), 0.1 mM non-essential amino acids (EuroClone), 1 mM sodium pyruvate (EuroClone), 50 μM β-mercaptoethanol (EuroClone) and 10 U/mL IL-2 (Miltenyi Biotec). Cells were stimulated for 4 h with 0.25 μM ionomycin and 10 ng/mL phorbol myristate acetate (PMA) and 2 hours with 10 μg/ml brefeldin A. A total of 1.5 × 106 cells were stained in PBS 1X/0.5% bovine serum albumin/2 mM EDTA for 10 min at 4°C with the following antibodies (Miltenyi Biotec): anti-CD3 Vioblue; anti-CD4-APC, anti-CD8-PE-Vio770, anti-CD62L-FITC and anti-CD49d-PE for T cell detection. The cells were then fixed and permealized using the Miltenyi Biotec Inside Stain Kit and intracellularly stained according to the manufacturer’s instructions with IFN-γ-PE or APC (Miltenyi Biotec). The CD8 + CD3+; CD3 + CD4+ cells were gated and then analyzed by flow cytometry for IFN-γ production and for the evaluation of CD62L and CD49d expression. Flow cytometry acquisition was performed on a MACSQuant instrument, and the data were analyzed with the MACSQuantify Software (Miltenyi Biotec).
Anti cd8 pe vio770
Manufactured by Miltenyi Biotec
Anti-CD8-PE-Vio770 is a fluorochrome-conjugated antibody that specifically binds to the CD8 antigen. It is designed for the identification and enumeration of CD8+ T cells in flow cytometric applications.
Automatically generated - may contain errors
Lab products found in correlation
Non essential amino acids (neaa), by Euroclone (1 mentions)
β mercaptoethanol, by Euroclone (1 mentions)
Anti cd3 vioblue, by Miltenyi Biotec (1 mentions)
Glutamine, by Euroclone (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Macsquantify software, by Miltenyi Biotec (1 mentions)
Penicillin, by Euroclone (1 mentions)
Streptomycin, by Euroclone (1 mentions)
Sodium pyruvate, by Euroclone (1 mentions)
Rpmi 1640, by Euroclone (1 mentions)
Anti cd4 apc, by Miltenyi Biotec (1 mentions)
Anti cd3 pe, by BioLegend (1 mentions)
Facsverse, by BD (1 mentions)
Fixable viability dye 450, by Thermo Fisher Scientific (1 mentions)
Collagenase 1, by Worthington (1 mentions)
Anti p24 fitc, by Beckman Coulter (1 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
3 protocols using anti cd8 pe vio770
1
Analyzing Antigen-Specific T Cell Responses
2
Quantifying CD4 T Cell Depletion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To compare infection kinetics and depletion in collagen and suspension cultures, cultures were first treated with collagenase I (100 U, Worthington) for 30 min at 37 °C to yield cell suspensions. Cells were washed in PBS and stained with fixable viability dye 450 (eBioscience, 1:1000 in PBS) for 30 min at 4 °C, were washed in MACS buffer (PBS, 2 mM EDTA, 0.5% inactivated FCS) and subsequently stained with anti-CD8-PE Vio770 (1:100, Miltenyi Biotec, 130-096-556) and anti-CD3-PE (1:100, biolegend, 317308) for 30 min at 4 °C. After washing, cells were fixed in 3% PFA/PBS for 90 min. To detect intracellular p24, cells were permeabilized and stained with anti-p24-FITC (1:100 KC57 Beckmann Coulter, 6604665) in 0.1% Triton-X-100/PBS for 30 min at 4 °C. Cells were washed in MACS buffer and for absolute cell quantification cell counting beads (biolegend) were added prior to analysis with a FACSVerse (BD) and FlowJo software (Tree Star). CD4 T cells were identified as CD3 positive/CD8 negative cells. Relative depletion was calculated by correlating the frequency of CD4 T cells in the respective sample to the frequency of CD4 T cells in T20 controls, which was set to 100%.
3
Multiparameter Flow Cytometry Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All cells were assessed for viability using a fixable viability dye (ebioscience eFluor506). Flow cytometric analysis of human and murine cells was performed using >50 antibodies and appropriate directly conjugated mouse, rat and hamster isotype controls. The clones . relevant to the majority of work are as follows: mouse anti-CD3-FITC (Miltenyi; REA641) anti-CD8a-PerCP (Miltenyi; 53-6.7) anti-CD28-PE (Miltenyi; 37.51) anti-KLRG1-APC (Miltenyi; 2F1). Human anti-CD3 APC-Cy7 (biolegend; HIT3a) anti-CD8-PE-Vio770 (Miltenyi; REA734) anti-CD28-eFluor450 (ebioscience; CD28.2) anti-KLRG1-AF488 (a generous gift from Professor Hanspeter Pircher, University of Freiburg) anti-CD57-Vioblue (Miltenyi; TB03) anti-CD244-FITC (biolegend; C1.7) anti-CX3CR1-PE (ebioscience; 2A9-1), and anti-IL-10-FITC (ebioscience; BT-10). For intracellular cytokine staining, cells were cultured with PMA (50 ng/ml), ionomycin (500 ng/ml), and monensin (3 µM; all from Sigma) for 4 h at 37C. Cells were stained for cell surface markers, then fixed and permeabilized in Cytofix/Cytoperm (BD) before intracellular detection of cytokines. Cells were acquired using a CyAn ADP (Beckman Coulter) flow cytometer and analysed using Summit software (software version 4.3; Beckman Coulter). Percentages and mean fluorescence intensity (MFI) were calculated against appropriate isotype controls.
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!