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Kavynfa

Manufactured by AnaSpec
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KAVYNFATM is a versatile instrument designed for the fast and accurate measurement of fluorescence signals. It utilizes a specialized detection system to quantify various fluorescent molecules or dyes in a sample. The core function of KAVYNFATM is to provide reliable and reproducible fluorescence measurements.

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Lab products found in correlation

Fixation permeabilization kit, by BD (2 mentions) Cd44 clone im7, by BioLegend (2 mentions) Thy1.1 clone ox 7, by BioLegend (2 mentions) Ifnγ clone xmg1, by BD (2 mentions) Thy1.2 clone 30 h12, by BioLegend (2 mentions) Cd8 clone 53 6, by BioLegend (2 mentions) I ab gp66 77 specific cd4 t cells, by BioLegend (2 mentions) Tnfα clone mp6 xt22, by Thermo Fisher Scientific (2 mentions) Cd4 clone gk1, by BioLegend (2 mentions) Lsrii flow cytometer, by BD (2 mentions) Lcmv gp33 41, by AnaSpec (1 mentions) Glngpdiykgvyqfksvefd, by AnaSpec (1 mentions) Ril 2, by Thermo Fisher Scientific (1 mentions) Murine recombinant cxcl10, by Thermo Fisher Scientific (1 mentions)

6 protocols using kavynfa

1

LCMV Infection and CD8+ T Cell Analysis

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Age- and sex-matched wildtype and knockout mice (6–8 weeks old) mice were infected intravenously with 2 × 106 plaque-forming units of LCMV clone 13 (provided by E. John Wherry, University of Pennsylvania). Thirty days after infection, mice were euthanized for the analysis of antigen-specific CD8 T cells in the spleen. In brief, splenocytes were stimulated with 3 μg/ml of LCMV GP33-41 peptide (KAVYNFATM; AnaSpec) for 6 h with monensin added during the last 2 hours. Cells were then subjected to staining of intracellular IFN-γ, IL-2, TNF-α and Granzyme B and flow cytometry analysis. To determine the virus titration in infected mice, spleen was collected and homogenized. The spleen homogenates were serially diluted and quantitated by plaque assay on Vero cell monolayers as previously described48 . In brief, Vero cells were plated in 6 well tissue culture plates and incubated overnight. Vero cells were then incubated with 10-fold dilutions of spleen lysates for 1 hour. The supernatant was aspirated and replaced with 4ml of 0.5% agarose in complete Medium 199. The plates were then incubated for 5 days and stained with 2ml of 0.5% agarose in complete Medium 199 containing 0.01% neutral red for overnight. Plaques were visually counted the next day.
Zhu L., Zhou X., Gu M., Kim J., Li Y., Ko C.J., Xie X., Gao T., Cheng X, & Sun S.C. (2022). Dapl1 controls NFATc2 activation to regulate CD8+ T cell exhaustion and responses in chronic infection and cancer. Nature cell biology, 24(7), 1165-1176.
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2

Flow Cytometric Analysis of Antigen-Specific T Cells

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For antigen-specific restimulation, 1 × 106 splenocytes were cultured for 6 hrs at 37°C in 5% CO2 in complete RPMI-1640 (10% HI-FBS, 55 μM beta-mercaptoethanol, 1x Pen/Strep and 1x L-Glut) supplemented with LCMV GP33–41 peptide KAVYNFATM (0.5 μg/mL, AnaSpec, cat. AS-61296) or left unstimulated.
For flow cytometric analysis, 1 × 106 splenocytes or LN cells were stained with antibodies specific for surface markers Thy1.2 (clone 30-H12, BioLegend), Thy1.1 (clone OX-7, BioLegend), CD8 (clone 53–6.7, BioLegend), CD4 (clone GK1.5, BioLegend), CD44 (clone IM7, BioLegend) and tetramers for either H2Db/GP33–43-specific CD8 T cells, H2Db/NP396–404-specific CD8 T cells or I-Ab/GP66–77-specific CD4 T cells (all from NIH Tetramer Core Facility). For intracellular staining, cells were processed using the Fixation/Permeabilization kit from BD Biosciences (BD Cytofix/Cytoperm, cat. 554714) following manufacturer’s instructions and stained with antibodies specific for IFNγ (clone XMG1.2, BD Biosciences) and TNFα (clone MP6-XT22, eBioscience). Samples were analysed on a BD Biosciences LSRII flow cytometer. Analysis of frequency and mean fluorescence intensity (MFI) was performed using FlowJo software. MFI was defined as the geometric mean of the fluorescence signal of interest for a given gated population.
Damo M., Fitzgerald B., Lu Y., Nader M., William I., Cheung J.F., Connolly K.A., Foster G.G., Akama-Garren E., Lee D.Y., Chang G.P., Gocheva V., Schmidt L.M., Boileve A., Wilson J.H., Cui C., Monroy I., Gokare P., Cabeceiras P., Jacks T, & Joshi N.S. (2020). Inducible de novo expression of neoantigens in tumor cells and mice with NINJA. Nature biotechnology, 39(1), 64-73.
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3

Activation of CD8+ T Cells

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Cells culture was set up in DMEM containing 10% FBS and 5 × 10−5 M 2-Mercaptoethanol. To activate CD8+ T cells, naïve T cells (CD62LhiCD44loCD8+) were stimulated with anti-CD3 or anti-CD3/28 antibodies for 72 h and harvested. CD8+ Teff used in this study were from naïve CD28WTCD8+ T cells stimulated by plate-bound anti-CD3/28 antibodies. To generate P14 CD8+ Teff cells, activate P14 CD8+ T cells (1 × 105 cell/mL) were activated by M2 antigenic peptide (LCMV gp33-41, KAVYNFATM, 0.08 nM, AnaSpec, Inc., San Jose, CA) presented by LPS-stimulated B6 B blast cells37 (link) (mitomycin C-treated38 (link), 5 × 105 cell/mL). At the end of the 3-day activation culture, the activated cells were washed and cultured in the presence of rhIL-2 (5 ng/mL) for 3–4 more days under 5% CO2.
Lai Y.P., Kuo L.C., Lin B.R., Lin H.J., Lin C.Y., Chen Y.T., Hsiao P.W., Chang H.T., Ko P.C., Chen H.C., Chang H.Y., Lu J., Ho H.N., Wu-Hsieh B.A., Kung J.T, & Chen S.C. (2021). CD28 engagement inhibits CD73-mediated regulatory activity of CD8+ T cells. Communications Biology, 4, 595.
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4

LCMV Antigen Peptides for Tetramer Assays

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Peptides for LCMV antigens GP33 (KAVYNFATM), GP276 (SGVENPGGYCL), NP396 (FQPQNGQFI), and GP61 (GLNGPDIYKGVYQFKSVEFD) were purchased from AnaSpec. PE-conjugated H2Db GP33-KAVYNFATM and I-Ab LCMV GP66-77 tetramers were provided by the NIH Tetramer Facility (Atlanta, GA).
Hu Y., Kim J.H., He K., Wan Q., Kim J., Flach M., Kirchhausen T., Vortkamp A, & Winau F. (2016). Scramblase TMEM16F terminates T cell receptor signaling to restrict T cell exhaustion. The Journal of Experimental Medicine, 213(12), 2759-2772.
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5

In vitro Expansion and Characterization of TCF1+ CD8+ T Cells

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In vitro TCF1+ cell culture was based on the protocol published in (Di Pilato et al., 2021 ). In brief, splenocytes from CD45.2 P14 GFP mice and CD45.2 Cxcr3−/− P14 mice were cultured at concentration of 107/ml in the presence of 1μg/ml of GP33–41 peptide (KAVYNFATM, AnaSpec). Alternatively, CD45.1 P14 mice and GFP+ CD45.2 Cxcr3−/− P14 cells were used for culturing. Twenty-four hours later, cells were harvested counted and re-plated at concentration of 106/ml with low amounts of rIL-2 (5ng/mL, Peprotech). Cells were cultured for 4 days, with a daily exchange of medium supplemented with fresh rIL-2. In some experiments, on day 3 of culture, 200ng/ml of murine recombinant CXCL10 (Peprotech) was added to the culture. On day 4, cells were controlled for the percentage of TCF1 and CXCR3 expression. For some experiments, cultured cells were i.v. transferred into Cl13-infected CD45.1 or CD45.2 C57BL6/J recipient mice to control their differentiation and localization within spleens.
Ozga A.J., Chow M.T., Lopes M.E., Servis R.L., Di Pilato M., Dehio P., Lian J., Mempel T.R, & Luster A.D. (2021). CXCL10 chemokine regulates heterogeneity of the CD8+ T cell response and viral set point during chronic infection. Immunity, 55(1), 82-97.e8.
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6

Flow Cytometric Analysis of Antigen-Specific T Cells

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For antigen-specific restimulation, 1 × 106 splenocytes were cultured for 6 hrs at 37°C in 5% CO2 in complete RPMI-1640 (10% HI-FBS, 55 μM beta-mercaptoethanol, 1x Pen/Strep and 1x L-Glut) supplemented with LCMV GP33–41 peptide KAVYNFATM (0.5 μg/mL, AnaSpec, cat. AS-61296) or left unstimulated.
For flow cytometric analysis, 1 × 106 splenocytes or LN cells were stained with antibodies specific for surface markers Thy1.2 (clone 30-H12, BioLegend), Thy1.1 (clone OX-7, BioLegend), CD8 (clone 53–6.7, BioLegend), CD4 (clone GK1.5, BioLegend), CD44 (clone IM7, BioLegend) and tetramers for either H2Db/GP33–43-specific CD8 T cells, H2Db/NP396–404-specific CD8 T cells or I-Ab/GP66–77-specific CD4 T cells (all from NIH Tetramer Core Facility). For intracellular staining, cells were processed using the Fixation/Permeabilization kit from BD Biosciences (BD Cytofix/Cytoperm, cat. 554714) following manufacturer’s instructions and stained with antibodies specific for IFNγ (clone XMG1.2, BD Biosciences) and TNFα (clone MP6-XT22, eBioscience). Samples were analysed on a BD Biosciences LSRII flow cytometer. Analysis of frequency and mean fluorescence intensity (MFI) was performed using FlowJo software. MFI was defined as the geometric mean of the fluorescence signal of interest for a given gated population.
Damo M., Fitzgerald B., Lu Y., Nader M., William I., Cheung J.F., Connolly K.A., Foster G.G., Akama-Garren E., Lee D.Y., Chang G.P., Gocheva V., Schmidt L.M., Boileve A., Wilson J.H., Cui C., Monroy I., Gokare P., Cabeceiras P., Jacks T, & Joshi N.S. (2020). Inducible de novo expression of neoantigens in tumor cells and mice with NINJA. Nature biotechnology, 39(1), 64-73.
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