OVA-specific CD8+ T cells from SDLN or spleen were analyzed by MHC-class I pentamer (257–264 SIINFEKL, Proimmune) staining. CTL activity was determined in vivo as described (36 (link)). Briefly, syngeneic spleen cells were stained with 5 or 0.3 μM CFSE, respectively. The latter fraction was pulsed with 10 μM CTL peptide (OVA 257–264 SIINFEKL) (37 (link)) or βGal 96–103, DAPIYTNV) (38 (link)) and combined with the first (non-pulsed) fraction. Of this mixture, 3 × 106 cells were injected i.v. into recipient mice. After 16 hrs, splenocytes and SDLN cells were analyzed by flow cytometry, and % specific lysis was calculated from the reduction of peptide-pulsed CFSEdim target cells relative to non-pulsed CFSEhi reference cells. Antigen-specific IFNγ-secreting CD8+ T cells were detected by ELISPOT technique, as described elsewhere (33 (link)). In brief, whole splenocytes or LN cells were cultured on ELISPOT plates coated with anti-mouse IFNγ mAb (clone: AN-18, eBioscience) in the presence or absence of CTL peptide (SIINFEKL or DAPIYTNV). After 24 hrs, cytokine spots were detected by use of biotinylated anti-mouse IFNγ mAb (clone: R46A2; Biolegend), followed by streptavidin-HRP and 3-amino-9-ethylcarbazol as a chromogenic substrate. Spots were counted manually from flatbed scans of duplicate wells.
Siinfekl
Manufactured by ProImmune
Sourced in United Kingdom
SIINFEKL is a synthetic peptide that serves as an antigenic epitope for MHC class I presentation. It is commonly used in immunological research and assays to study T cell responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse ifn γ mab, by Thermo Fisher Scientific (1 mentions)
Golgiplug, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Perm wash, by BD (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Pe labeled h 2kb siinfekl, by ProImmune (1 mentions)
3 protocols using siinfekl
1
In Vivo Cytotoxicity Assay for CD8+ T Cells
2
Intracellular Cytokine Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For intracellular cytokine staining, 1x106 cells per well were plated on flat-bottom tissue-culture-treated 96-well plates. Cells were stimulated for 5 hours at 37C in the presence of human recombinant IL-2 (10 U/well), and brefeldin A (1 μl/ml, GolgiPlug, BD Biosciences), with one of the following peptides: SIINFEKL, NP366, PA224 (thinkpeptides®, ProImmune Ltd. Oxford, UK) at 0.1ug/ml, or without peptide. After stimulation, cells were stained for surface markers, and then processed with Cytofix/Cytoperm kit (BD Biosciences, Franklin Lakes, NJ). Permeabilized cells were transferred to FACS buffer for acquisition, while surface-stained cells were fixed with 2% paraformaldehyde in PBS for 20 minutes, then transferred to FACS buffer. All samples were acquired on FACSCalibur, LSR II, or LSRFortessa (BD Biosciences) analytical flow cytometers. Data were analyzed with FlowJo software (TreeStar, Ashland, OR).
3
Characterization of Antigen-Specific CD8 T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenocytes were prepared as described above. For analysis of SIINFEKL-specific CD8 T cell proliferation, the cells were Fc receptor blocked with anti-CD16/32 and stained with fluorescent anti-CD8 and anti-CD44 Abs and PE-labeled H2Kb-SIINFEKL (ProImmune, Oxford, U.K.). For analysis of intracellular cytokine production, the cells were incubated overnight with 10 μg/ml OVA and 2.5 μg/ml brefeldin A (Sigma) for the last 4 h. After washing and Fc receptor blocking, the cells were stained with anti-CD4, anti-CD8, and anti-CD44; fixed in 4% paraformaldehyde; permeabilized in Perm/Wash (BD Biosciences); and stained with anti–IFN-γ and anti–TNF-α Abs. All stainings were done in cold PBS supplemented with 2% FBS and protected from light. All staining Abs were from eBioscience or BD Pharmingen. The stained cells were acquired on BD FACSCanto II (BD Biosciences) and analyzed with FlowJo (FlowJo, Ashland, OR).
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!