Fluorescent target array killing assay: A multiplex cytotoxic T‐cell assay to measure detailed T‐cell antigen specificity and avidity in vivo

Abstract

Here we describe a multiplex, fluorescence‐based, in vivo cytotoxic T‐cell assay using the three vital dyes carboxyfluorescein diacetate succinimidyl ester, cell trace violet, and cell proliferation dye efluor 670. When used to label cells in combination, these dyes can discriminate >200 different target cell populations in the one animal due to each target population having a unique fluorescence signature based on fluorescence intensity and the different emission wavelengths of the dyes. This allows the simultaneous measurement of the in vivo killing of target cells pulsed with numerous peptides at different concentrations and the inclusion of many replicates. This fluorescent target array killing assay can be used to measure the fine antigen specificity and avidity of polyclonal cytotoxic T‐cell responses in vivo, immunological parameters that were previously impossible to monitor. © 2012 International Society for Advancement of Cytometry

Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution

“…While all five have been shown to be able to track proliferation with varying degree of success in primary lymphocytes [22,25], we have noted from our data and that of other groups the overall ''quality'' of peak resolution with lipophilic dyes is particularly poor [11,26] even after sorting to reduce fluorescence spread [18]. Our method was developed to specifically evaluate how any candidate dye for fluorescence proliferation tracking by dye dilution would perform in vitro, with particular emphasis on situations where the limits for peak resolution have been exceeded due to the inherent heterogeneity of the cell type.…”

Antigen presenting capacity of murine splenic myeloid cells

“…Targeted cell lysis was measured using a fluorescent target array developed by Quah et al [35]. The experimental protocol is summarised in Fig.…”

Use of an In Vivo FTA Assay to Assess the Magnitude, Functional Avidity and Epitope Variant Cross-Reactivity of T Cell Responses Following HIV-1 Recombinant Poxvirus Vaccination

“…Peptides included the MHC-I-binding peptides, SPYAAGYDL (F2L, an L d -restricted vaccinia virus (VV) epitope), SP G AAGYDL (F2L mut, a modified vaccinia Ankara virus epitope homologous to F2L), AMQMLKETI (HIV Gag, an immunodominant K d -restricted HIV Gag epitope [24]), AMQMLK D TI (HIV Gag mut, a HIV Gag subtype C variant of HIV Gag [25]), VGPTPVNII (HIV Pol, a D d -restricted HIV pol epitope [26]), RGPGRAFVTI (HIV envelop (Env), a D d -restricted HIV env epitope [27], and AMQMLEKTI (HIV neg, a modified HIV gag epitope that serves as a negative control [28]) and the MHC-II-binding peptide PVGEIYKRWIILGLN (Gag T H , a H-2 d -restricted HIV Gag epitope [24]). Peptides were synthesized at the Biomolecular Resource Facility, John Curtin School of Medical Research, ANU.…”

“…The % specific killing and T cell help was assessed as previously described [11], [12], [28]. Briefly, for the CTL responses, the number of cells in each FTA cell cluster was calculated using FlowJo software and the % specific killing values were then generated using the following formula: …”