Journal: STAR Protocols

Article Title: Protocol for isolating stromal cells from lymphoid tissue for performing scRNA-seq

doi: 10.1016/j.xpro.2026.104501

Figure Lengend Snippet: Composition of cells recovered after each digestion step Collected cells were analyzed after each digestion step to determine cell phenotypes, numbers and viability. When performing transcriptomics analysis all digestion fractions will be combined. (A) Percentages of immune cells (CD45 + cells) and stromal cells (CD45 - cells) extracted from each digestion step analyzed via flow cytometry. (B) Representative viability images of each digestion step acquired on automated cell counter (LUNA 7-FX™) where red indicates dead cells and green live cells. (C) Immunofluorescence images of cells stained after each digestion step for CD45 (yellow) and Nuclei (blue). Representative Cytospin slides. Red arrows showing examples of CD45 - cells. Scale bars indicate 100 μm for main images and 10 μm for zoom. (D) After each digestion step the (i) percentage of live cells, (ii) number of live cells after each digestion measured by automated cell counting and (iii) percentage of immune (gray) and stromal cells (red) after each digestion fraction. (E) Combined cells from all digestions showing percentage and number of cells from each lymph node. (F) Representative gating strategy for lymph node cells showing stromal cell percentages. (G–L) Number of (G) immune cells, (H) stromal cells, (I) fibroblastic reticular cells (FRC), (J) lymphatic endothelial cells (LEC), (K) blood endothelial cells (BEC) and (L) double negative cells (DNC).

Article Snippet: CD45 antibody, anti-mouse, Biotin (Dilutions in 1:50) , Miltenyi Biotec , Cat# 130-124-209, RRID: AB_2819580.

Techniques: Transcriptomics, Flow Cytometry, Immunofluorescence, Staining, Cell Counting

Journal: STAR Protocols

Article Title: Protocol for isolating stromal cells from lymphoid tissue for performing scRNA-seq

doi: 10.1016/j.xpro.2026.104501

Figure Lengend Snippet: Cell selection using automated magnetic cell sorting (A) Cells were stained with CD45-biotin and CD31-biotin and sorted using autoMACS® Pro Separator. (B) Number of cells before staining for autoMACS® separation (step 11), and number of cells recovered from positive selection (CD45 + and CD31 + cells) and negative selection (CD45 - and CD31 - cells) in step 23. Each dot represents combined numbers from inguinal, axillary and brachial lymph nodes from 6 mice. Colors indicate biological replicates. (C) Percentages of cells after separation compared to the pre-staining cell count performed in step 11. (D) Purity check of separated cells using flow cytometry and staining for CD45 and CD31. (E) Overlay plots of positive selection (blue) and negative selection (red). (F) Percentage of CD45 + , CD45 - and CD31 + cells recovered (∗∗∗∗ p value < 0.0001, ∗ p value < 0.05). (G) Final viability check of positive and negative selected cells acquired just before performing scRNA-sequencing analysis.

Article Snippet: CD45 antibody, anti-mouse, Biotin (Dilutions in 1:50) , Miltenyi Biotec , Cat# 130-124-209, RRID: AB_2819580.

Techniques: Selection, FACS, Staining, Cell Characterization, Flow Cytometry, Sequencing

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of a hematopoietic stem cell-delivered cell-penetrating frataxin in Friedreich’s ataxia models

doi: 10.1016/j.xcrm.2026.102803

Figure Lengend Snippet: LV-FXN gene therapy does not affect the engraftment and lineage commitment of HSPCs and deposits FXN protein in FRDA-relevant tissues (A) Overview of the transplantation experiment. Lineage-negative cells isolated from LY5.1 mice were transduced with LV-FXN at a multiplicity of infection (MOI) of 20 and transplanted into lethally irradiated LY5.2 recipient mice. Three months after transplantation, hematopoietic organs were analyzed by FACS, and frataxin protein levels were measured in the spleen, brain, heart, muscle, liver, and kidney by mass spectrometry. (B–D) Percentage of CD45.1 (donor-derived) and CD45.2 (recipient-derived) cells in peripheral blood, bone marrow (BM), and spleen of mice transplanted with mock-untransduced ( n = 3) or LV-FXN-transduced cells ( n = 4) (upper), along with the lineage composition within the CD45.1 and CD45.2 compartments (lower). (E) Levels of human mature frataxin (ng per mg of total protein; mean ± SD) in the spleen of mice transplanted with mock-untransduced cells (mouse #304) or LV-FXN-transduced cells (mice #306, #307, and #310). (F) Total frataxin levels (ng per mg of total protein; mean ± SD) in the indicated organs of mice transplanted with mock-transduced cells (mouse #304) or LV-FXN- transduced cells with >1 vector copy number (VCN) (mice #307 and #310). ND = not determined.

Article Snippet: Anti-mouse CD45.2 FITC , Miltenyi , Cat# 130-102-458; RRID:AB_2660717.

Techniques: Transplantation Assay, Isolation, Transduction, Infection, Irradiation, Mass Spectrometry, Derivative Assay, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of a hematopoietic stem cell-delivered cell-penetrating frataxin in Friedreich’s ataxia models

doi: 10.1016/j.xcrm.2026.102803

Figure Lengend Snippet: LV-FXN gene therapy does not affect the engraftment and lineage commitment of HSPCs and deposits FXN protein in FRDA-relevant tissues (A) Overview of the transplantation experiment. Lineage-negative cells isolated from LY5.1 mice were transduced with LV-FXN at a multiplicity of infection (MOI) of 20 and transplanted into lethally irradiated LY5.2 recipient mice. Three months after transplantation, hematopoietic organs were analyzed by FACS, and frataxin protein levels were measured in the spleen, brain, heart, muscle, liver, and kidney by mass spectrometry. (B–D) Percentage of CD45.1 (donor-derived) and CD45.2 (recipient-derived) cells in peripheral blood, bone marrow (BM), and spleen of mice transplanted with mock-untransduced ( n = 3) or LV-FXN-transduced cells ( n = 4) (upper), along with the lineage composition within the CD45.1 and CD45.2 compartments (lower). (E) Levels of human mature frataxin (ng per mg of total protein; mean ± SD) in the spleen of mice transplanted with mock-untransduced cells (mouse #304) or LV-FXN-transduced cells (mice #306, #307, and #310). (F) Total frataxin levels (ng per mg of total protein; mean ± SD) in the indicated organs of mice transplanted with mock-transduced cells (mouse #304) or LV-FXN- transduced cells with >1 vector copy number (VCN) (mice #307 and #310). ND = not determined.

Article Snippet: Anti-mouse CD45.1 PE , Miltenyi , Cat# 130-102-499; RRID:AB_2660704.

Techniques: Transplantation Assay, Isolation, Transduction, Infection, Irradiation, Mass Spectrometry, Derivative Assay, Plasmid Preparation