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.

Keywords: Ascorbate; Vitamin C.

Fig. 1

Development of the Single-cell Ascorbate Level Sensing Assay (SALSA). (A) Biochemical assay of AA detection. AA is chemically oxidized with 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) into dehydroascorbate (DHA), which then reacts with the diaminobenzene structure of o-phenylenediamine (OPDA) to generate a fluorescent product. The diamine group is labeled in a red circle. (B) OPDA and DAF-2 share a diaminobenzene group. The chemical structure of DAF-2 is shown, and the diamine group is highlighted. (C–D) Chemical structures of products for DAF-2 reaction with (C) nitric oxide (DAF-2 triazole or DAF-2T) or (D) DHA (DAF-2-DHA). (E) An overview of experimental design. HEK293T cells were cultured with or without 1 mM AA for 24 h. (F) Intracellular AA level was analyzed using the biochemical assay as in (A). (G) Increased green fluorescence signal only in the presence of TEMPOL and AA. Cells were cultured as in (E), labeled with the cell-permeable DAF-2-diacetate (DAF-2-DA), and treated with or without TEMPOL (TP) as indicated. Cells were analyzed by flow cytometry using the FITC channel at 515–545 nm. (H) The principle of the Single-cell Ascorbate Level Sensing Assay, or SALSA. Cells were labeled with DAF-2-DA at 37 °C for 10 min and then treated with TEMPOL at 25 °C for 10 min. Cells were analyzed using flow cytometry. (I–J) Optimization of SALSA dye. Cells cultured with or without AA were labeled with the indicated concentrations of cell-permeable DAF-2 or DAF-FM diacetates. (I) The median fluorescence intensity (MFI) and (J) the MFI ratio between cells cultured with and without AA were calculated for each condition. (K–L) Optimal DAF-2 labeling time. Cells were labeled with DAF-2-DA for either 10 or 20 min. Data were analyzed using (F) Student's t-test where ∗∗∗P ≤ 0.001; or (I–L) two-one-way ANOVA followed by Tukey's multiple comparisons test. Bars not sharing the same letter are significantly different (P ≤ 0.05). Representatives of at least two independent experiments are shown.

Fig. 2

Unexpected spectral red-shift significantly enhances SALSA sensitivity. HEK293T cells were cultured with or without AA for 24 h and analyzed by SALSA. (A–B) TEMPOL- and AA-dependent signal has significant spillover from FITC (SALSA^Verde) to PE (SALSA^Roja) channels. (A) FITC/PE signals from the Unstained (blue), Mock (gray), and AA (orange) groups were overlayed. Left, original signals without compensation. Right, signals in the two channels were compensated using the fluorescence from the Mock group. (B) Data from (A) are depicted as histograms. Signals before (Uncompensated) and after compensation (Compensated) for the PE (SALSA^Roja) channel are shown. (C) The primary SALSA signal derived from TEMPOL and AA is red-shifted. Data was compensated as in (A) and (B), and the MFI ratio (Fold vs Mock; MFI_AA/MFI_Mock) between cells cultured with and without AA was plotted for each open channel on a three-laser BD FACSCanto II. (D–E) Equipment-dependent enhancement of SALSA sensitivity. Cells were analyzed using SALSA with a five-laser BD FACSymphony A3. (D) Signals before and after compensation were shown for the BB515 (FITC equivalent; SALSA^Verde) and BB630 (PE equivalent excited by the 488 nm blue laser; SALSA^Roja). (E) The MFI ratios were calculated as in (C) for all available channels. (F–J) SALSA is quantitative and directly correlated with intracellular AA concentration. HEK293T cells were cultured with varying concentrations of AA for 24 h. (F) Intracellular AA is quantified using the biochemical assay described in Fig. 1a. Cells were analyzed using SALSA, and the MFIs for BB515 (SALSA^Verde) and BB630 (SALSA^Roja) are shown in (G) and (I). MFI was plotted against the intracellular AA concentration in (H) and (J). Linear regression was calculated, and the coefficient of determination (R²) is shown. (K–O) Establishment of standard curve. (K) HEK293T cells were permeabilized using streptolysin O (SLO), incubated with the indicated concentration of “spike-in” AA, sealed, followed by SALSA. (L–O) Representative histograms and graphs showing the linear correlation between SALSA signals and the spike-in AA levels. The dotted lines indicate the background levels at 1. Data were analyzed using two-one-way ANOVA followed by Tukey's multiple comparisons test. Bars not sharing the same letter are significantly different (P ≤ 0.05). Representatives of at least two (A–J) and four (K–O) independent experiments are shown.

Fig. 3

SALSA can specifically detect ascorbate with minimal NO interference. (A) AA has no significant effect on macrophage activation. Top, Murine macrophage cell line Raw264.7 was stimulated with LPS (50 ng/mL) and IFN-γ (10 ng/mL) overnight with or without AA (1 mM). Bottom, histograms showing the expression of CD80, an activation marker, and iNOS, an enzyme for NO production. (B) iNOS inhibition abolishes NO production. Cells were cultured as in (A) with or without 20 μM 1400W, a selective and potent iNOS inhibitor. Nitrite ion, a proxy for NO production, was detected using the Griess assay. (C–E) SALSA enables specific detection of AA in the presence of NO. Unstimulated (top) and stimulated (bottom) Raw264.7 cells were analyzed using SALSA with a BD FACSymphony. Histograms for BB515 (SALSA^Verde) and BB630 (SALSA^Roja) channels (C) and the MFI quantification (D–E) are shown. (F–H) SALSA can detect AA in the presence of NO. Stimulated Raw264.7 cells were treated with AA and 1400W as indicated and analyzed using SALSA. Histograms for BB515 (SALSA^Verde) and BB630 (SALSA^Roja) channels (F) and the MFI quantification (G–H) are shown. (I) iNOS inhibition and NO quenching significantly improved SALSA by eliminating interference from NO. Raw264.7 cells were cultured with AA and stimulated as indicated for 24 h. iNOS inhibitor 1400W was added throughout stimulation and NO quencher PTIO was added 30 min before SALSA. SALSA^Verde (BB515; top panel) and SALSA^Roja (BB630; bottom panel) signals are shown. Data were analyzed using two-one-way ANOVA followed by Tukey's multiple comparisons test. Bars not sharing the same letter are significantly different (P ≤ 0.05). Representatives of at least two independent experiments are shown.

Fig. 4

Identification of SVCT2 as the major ascorbate transporter in HEK293T cells using SALSA and CRIPSR. SVCT1 (SLC23A1) and SVCT2 (SLC23A2) were targeted in HEK293T cells using lentivirus expressing Cas9 and sgRNA. (A) Confirmation of sgRNA targeting efficiency. DNA flanking sgRNA-targeting sites were amplified by PCR. Amplicons were sequenced using Sanger, and the results were analyzed using TIDE assay (Tracking of Indels by Decomposition) to calculate the indel frequency. Estimated targeting efficiencies for SVCT1 and SVCT2 were at least 71.7 % and 69.2 %, respectively. (B) Scheme for the internally controlled SALSA. Positive control (blue) and CRISPR-targeted cells (red) were cultured with AA for 24 h. Positive control and cells from each condition were labeled with antibodies against β2M, a universal antigen, conjugated with different fluorescence. The two groups of cells were mixed and underwent SALSA in the same reaction for increased consistency. (C) Representative FACS plots show the distinction between the mixed populations. (D–E) Mutation of SVCT2 significantly decreased intracellular AA. Histograms (D) and MFI quantifications (E) for the SALSA^Roja signal (PE) are shown. Data was analyzed using paired Student's t-test. ∗P ≤ 0.05; ns, not significant. Except for (A), representatives of four independent experiments are shown. Note that the results for the other two sgRNAs were similar (not shown).

Fig. 5

SALSA reveals heterogeneous ascorbate levels among blood cells. (A) The experiment setup. Gulo-deficient mice, which cannot synthesize AA, were supplied with (AA^Sufficient) or without AA (AA^Deficient) for three weeks. (B–D) Cells from blood, thymus, and spleen in AA^Sufficient and AA^Deficient mice were harvested, counted (thymus and spleen), and analyzed using SALSA (C–D). Gray areas indicate background signals from DAF-2 without TEMPOL-mediated AA oxidation. (E–G) Analysis of AA level in developing T cells. Thymocytes from AA-sufficient and -deficient mice were analyzed using SALSA. SALSA reaction without TEMPOL was shown as the background. (F) Gating strategy for thymocytes. DN, CD4 CD8 double negative; CD4SP, single positive. (G) Intracellular AA levels were inversely correlated with maturation stages. (H–K). Impact of AA deficiency on thymic populations. (H–I) Representative FACS plots for total thymocytes (H) and CD4SP cells (I). (J–K) The percentage of subsets was quantified and analyzed using two-tailed Student's t-test. ∗∗∗P ≤ 0.001; ∗∗P ≤ 0.01; ∗P ≤ 0.05; ns, not significant. Representative data were shown from 3 independent experiments.