act  (Mankind Pharma Ltd)

Journal: Cell Reports Methods

Article Title: Single-cell assessment of iron content in primary human T cells using laser ablation inductively coupled plasma mass spectrometry

doi: 10.1016/j.crmeth.2026.101343

Figure Lengend Snippet: LA-ICP-MS provides the required sensitivity to assess endogenous iron content in individual primary T cells (A) Experimental set up, briefly, flow-sorted CD3 + T cells were counted and plated in MS buffer onto poly-L-lysine-coated removable 8-well chambers. Cells were gently centrifuged to promote adherence. All remaining buffer was removed by aspiration, and cells were subsequently washed by metal trace water. Cells were visually selected for laser ablation and iron content assessed by ICP-MS. (B and C) Visualization of adherent T cells by light microscopy (upper image) and on LA-ICP-MS connected system (lower image) selected cells for ablation are marked. (D) Complementary techniques for obtaining blank measurements from the slide in areas without cells. Blank method 1 (left), obtaining blank measurements independently of other measurements. Blank method 2 (right), involves taking blank measurements throughout the ICP-MS run and confirms the slide is free external analyte contamination. Highlighted throughout the run are blank measurements obtained throughout one continuous ICP-MS analysis run. (E) Experimental workflow for iron loading. Cells were FACS sorted into pure CD3 + T cell populations and cultured for 40 h, with 100 μM of iron citrate added in the last 10 h of culture prior to LA-ICP-MS analysis. (F) Intracellular iron (fg Fe) per CD3 + sorted T cells for 3 donors, with table showing mean Fe per single cell (horizontal line). Each dot represents iron content per single cell. nD1 = 89, nD2 = 104, nD3 = 99. (G) Single-cell iron quantification for fresh and frozen unstimulated CD3-sorted T cells without and with iron loading (100 μM). The bar represents mean fg of Fe per single cell. Statistical significance was determined using a multiple Mann-Whitney test; p value written above. Each symbol represents a single cell from D8; n fresh,-Fe = 91, n fresh,+Fe = 112, n frozen,-Fe = 49, n frozen,+Fe = 99. (H) Pooled data from two donors (D1 and D13) of single-cell iron quantification (fg) of fixed (mean fg per cell = 1.41) and unfixed (mean fg per cell = 1.59). Each symbol represents the mean intracellular iron from an individual donor; n = 2 donors.

Article Snippet: Subsequently, cells were cultured in complete RPMI 1640 medium (cRPMI) supplemented with 2mM L-glutamine, 0.1mM non-essential amino acids, 10mM HEPES buffer, 1mM sodium-pyruvate and 10% FCS and activated using T cell Trans-Act which delivers T cell activation via CD3 and CD28 (Miltenyi) for 2 days in the presence of 100IU/mL rhIL-2 (Aldesleukin).

Techniques: Light Microscopy, Cell Culture, Single Cell, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: Single-cell assessment of iron content in primary human T cells using laser ablation inductively coupled plasma mass spectrometry

doi: 10.1016/j.crmeth.2026.101343

Figure Lengend Snippet: Accuracy of LA-ICP-MS measurements as validated by bulk ICP-MS (A) Schematic of the workflow analysis for bulk ICP-MS. T cells were counted and digested in 65%–69% HNO 3 before being resuspended in reverse osmotic pure H 2 O and split into triplicates. Samples were run on the NexION5000. (B–D) Metal quantification of iron (Fe), zinc (Zn), and magnesium (Mg) quantified in pg/million cells in activated CD3 T cells for three healthy donors ( n = 3). Tables below represent mean fg/cell converted from pg/million cells for each donor. BLQ, below the limit of quantification.

Article Snippet: Subsequently, cells were cultured in complete RPMI 1640 medium (cRPMI) supplemented with 2mM L-glutamine, 0.1mM non-essential amino acids, 10mM HEPES buffer, 1mM sodium-pyruvate and 10% FCS and activated using T cell Trans-Act which delivers T cell activation via CD3 and CD28 (Miltenyi) for 2 days in the presence of 100IU/mL rhIL-2 (Aldesleukin).

Techniques:

Journal: Cell Reports Methods

Article Title: Single-cell assessment of iron content in primary human T cells using laser ablation inductively coupled plasma mass spectrometry

doi: 10.1016/j.crmeth.2026.101343

Figure Lengend Snippet: LA-ICP-MS analysis identifies difference in iron content between T cell subsets (A) Representative figure of experimental workflow. PBMCs were FACS sorted for CD3 + , CD4 + , and CD8 + T cells and cultured for 40 h in complete RPMI. Intracellular iron was quantified by LA-ICP-MS. (B) Intracellular iron (fg Fe) per cell CD4 + and CD8 + sorted T cells for D1 and D3 (shown in F). Statistical significance was determined using a multiple Mann-Whitney test; p value written above. Each symbol represents a single cell, nD1, CD4 = 63, nD1, CD8 = 122, nD3, CD4 = 104, and nD3, CD8 = 118, and mean values are represented by black horizontal line.

Article Snippet: Subsequently, cells were cultured in complete RPMI 1640 medium (cRPMI) supplemented with 2mM L-glutamine, 0.1mM non-essential amino acids, 10mM HEPES buffer, 1mM sodium-pyruvate and 10% FCS and activated using T cell Trans-Act which delivers T cell activation via CD3 and CD28 (Miltenyi) for 2 days in the presence of 100IU/mL rhIL-2 (Aldesleukin).

Techniques: Cell Culture, MANN-WHITNEY, Single Cell

Journal: Cell Reports Methods

Article Title: Single-cell assessment of iron content in primary human T cells using laser ablation inductively coupled plasma mass spectrometry

doi: 10.1016/j.crmeth.2026.101343

Figure Lengend Snippet: Single-cell analysis reveals changes in iron content upon T cell activation (A) Representative figure of experimental workflow. PBMCs were FACS sorted for CD3 + T cells and cultured for 40 h in complete RPMI with CD3/CD28 stimulation. Subsequently intracellular iron was quantified by LA-ICP-MS. (B) Intracellular iron quantification per single cell for 6 donors, for stimulated (blue) and unstimulated (purple) CD3 + T cells. Data are shown from two independent experiments. Statistical significance was determined using a multiple Mann-Whitney test, significant p values written above. Donors 4–7 and donors 8–9 were analyzed in two independent experiments. Each individual symbol represents an single cell analyzed and mean values are represented by black horizontal line; nD4,unstim = 101, nD4,stim = 62, nD5,unstim = 110, nD5,stim = 94, nD6,unstim = 107, nD6,stim = 103, nD7,unstim = 99, nD7,stim = 100, nD8,unstim = 91, nD8,stim = 114, nD8,unstim = 99, nD9,stim = 91. (C) Summery data table of mean Fe (fg) per single for stimulated and unstimulated CD3 + sorted T cells. (D) Relative size of stimulated and unstimulated T cells, surface expression of transferrin receptor, CD71, in stimulated (blue) and unstimulated (purple) T cells, and representative histogram of CD71 surface expression in stimulated and unstimulated T cells by flow cytometry, normalized to the mode. Shown for two independent experiments for the donors analyzed in (C), n = 6 donors. Mean values are represented by a horizontal black line throughout figure.

Article Snippet: Subsequently, cells were cultured in complete RPMI 1640 medium (cRPMI) supplemented with 2mM L-glutamine, 0.1mM non-essential amino acids, 10mM HEPES buffer, 1mM sodium-pyruvate and 10% FCS and activated using T cell Trans-Act which delivers T cell activation via CD3 and CD28 (Miltenyi) for 2 days in the presence of 100IU/mL rhIL-2 (Aldesleukin).

Techniques: Single-cell Analysis, Activation Assay, Cell Culture, Single Cell, MANN-WHITNEY, Expressing, Flow Cytometry