{"doi":"10.1016/j.redox.2025.103823","title":"SALSA: a novel flow cytometry assay to detect ascorbate at the single-cell level","abstract":"Ascorbate (AA) is an essential antioxidant and enzymatic cofactor with emerging roles in epigenetic regulation, redox biology, and immune function. However, single-cell quantification of intracellular AA has remained technically challenging. Here, we present SALSA (Single-cell Ascorbate Level Sensing Assay), a novel flow cytometry-based method that enables sensitive, specific detection of intracellular AA at the single-cell level. Inspired by the mechanism of the in vitro AA assay, we identified 4,5-diaminofluorescein (DAF-2), a common nitric oxide (NO) probe, as a selective AA reporter. We showed that the chemical oxidation of AA into dehydroascorbic acid (DHA) facilitated its reaction with DAF-2 to form a highly fluorescent product. Surprisingly, the DAF-2-DHA adduct exhibits a red-shifted emission spectrum distinguishable from those of DAF-2 alone or its NO-reactive product. This spectral shift enables the differentiation of signals into two channels, SALSA Verde (green) and SALSA Roja (red-orange), with SALSA Roja offering superior sensitivity and minimal NO interference. SALSA is quantitative, with a strong linear correlation between signal intensity and intracellular AA concentration. Using SALSA and CRISPR, we identified SVCT2 as the major AA transporter in a human cell line model. Applying SALSA to immune profiling revealed previously unappreciated heterogeneity in AA levels across immune subsets and developmental stages. Together, these findings establish SALSA as a robust and accessible method for probing AA dynamics at single-cell resolution, with broad potential applications in redox biology, immunology, and metabolism.","journal":"Redox Biology","year":2025,"id":546175,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9557,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1438306,"name":"W. Chou","orcid":null,"position":1,"is_corresponding":false},{"id":817933,"name":"Heng-Yi Chen","orcid":"0000-0001-9283-6144","position":2,"is_corresponding":false},{"id":1438307,"name":"Dena Mossad","orcid":null,"position":3,"is_corresponding":false},{"id":1418785,"name":"Michael Hsu","orcid":"0000-0003-3852-1461","position":4,"is_corresponding":false},{"id":1038582,"name":"Bridget A. Bowman","orcid":null,"position":5,"is_corresponding":false},{"id":1218626,"name":"Kartick Patra","orcid":"0000-0002-5636-7739","position":6,"is_corresponding":false},{"id":396741,"name":"Huan Zhang","orcid":"0000-0002-1096-4255","position":7,"is_corresponding":false},{"id":818599,"name":"Fang-Yun Lay","orcid":null,"position":8,"is_corresponding":false},{"id":1437923,"name":"Frederick A. Villamena","orcid":"0000-0002-7330-8431","position":9,"is_corresponding":false},{"id":78715,"name":"Gang Xin","orcid":null,"position":10,"is_corresponding":false},{"id":447626,"name":"Richard S. Bruno","orcid":"0000-0002-6772-2038","position":11,"is_corresponding":false},{"id":360755,"name":"Mark Levine","orcid":"0000-0003-2800-8728","position":12,"is_corresponding":false},{"id":1437924,"name":"Jiangjiang Zhu","orcid":"0000-0002-9156-9870","position":13,"is_corresponding":false},{"id":456774,"name":"Fernanda O. Novais","orcid":"0000-0002-9491-0384","position":14,"is_corresponding":false},{"id":304129,"name":"Chan‐Wang Jerry Lio","orcid":"0000-0003-3876-6741","position":15,"is_corresponding":false},{"id":1312510,"name":"Bella Lee","orcid":"0000-0002-4848-7829","position":0,"is_corresponding":true}],"reference_count":48,"raw_metadata":null,"created_at":"2026-07-19T02:53:32.285899Z","pmid":"40854263","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}