Journal: STAR Protocols

Article Title: Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging

doi: 10.1016/j.xpro.2022.101828

Figure Lengend Snippet: Sample preparation and staining panel—Mouse HSC

Article Snippet: APC-Cy7 Hamster Anti-Mouse CD48; Clone HM48-1 , BD Biosciences , Cat#561242.

Techniques: Sample Prep, Staining, Positive Control

Journal: STAR Protocols

Article Title: Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging

doi: 10.1016/j.xpro.2022.101828

Figure Lengend Snippet: FACS gating strategy to sort MMP-Low or -High CD48-LSK CD150 + mouse HSCs (A) Determine Lin - CD48 - gate (Gate 1). Flow cytometry APCCy7 contour plots of total BM (Lin/CD48)-APCCy7 positive staining control (i), and Lin - enriched BM full stain sample (ii). (B) Flow cytometry gating scheme towards CD48 - LSKCD150 + from Lin - enriched mouse BM. i, ii, ii, iv, gating debris - (i), doublets - (ii, iii), DAPI - (iv) single viable cells. v, vi, vii, sequential gating of Lin - CD48 - BM (Gate 1; v), Sca-1 + c-Kit + (Gate 2;vi), CD150 + (Gate 3; vii). viii, TMRE Flow histogram of CD48 - LSKCD150 + HSCs. Set bottom 25% MMP-Low and top 25% MMP-High gates.

Article Snippet: APC-Cy7 Hamster Anti-Mouse CD48; Clone HM48-1 , BD Biosciences , Cat#561242.

Techniques: Flow Cytometry, Staining

Journal: STAR Protocols

Article Title: Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging

doi: 10.1016/j.xpro.2022.101828

Figure Lengend Snippet: Validation of the specificity and responsiveness of TMRE staining to changes in MMP level using CCCP and oligomycin Flow cytometry histogram of TMRE for CD48 - LSKCD150 + HSCs treated with CCCP (100 μM, red), or oligomycin (2 μg/mL, blue), or untreated (DMSO, 10 min, black). Depolarized and hyperpolarized TMRE gates were determined according to the peak separation on TMRE profile of CCCP and oligomycin treated samples respectively. Only 0.96% or 1.44% untreated HSCs are depolarized or hyperpolarized. TMRE intensity of 97.6% of cells are within physiological range.

Article Snippet: APC-Cy7 Hamster Anti-Mouse CD48; Clone HM48-1 , BD Biosciences , Cat#561242.

Techniques: Staining, Flow Cytometry

Journal: STAR Protocols

Article Title: Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging

doi: 10.1016/j.xpro.2022.101828

Figure Lengend Snippet:

Article Snippet: APC-Cy7 Hamster Anti-Mouse CD48; Clone HM48-1 , BD Biosciences , Cat#561242.

Techniques: Recombinant, Cell Isolation, Selection, Software

Journal: STAR Protocols

Article Title: Isolation of human and murine hematopoietic stem cells for DNA damage and DNA repair assays

doi: 10.1016/j.xpro.2021.100846

Figure Lengend Snippet: Strategies for enrichment of HSPC (A) Human Lin - cells can be enriched using a magnetic stand. Left. Cells are incubated with the progenitor cell enrichment cocktail followed by incubation with magnetic particles and incubation in the magnetic stand. Right. Lin + cells bound by the antibodies of the enrichment cocktail and by the magnetic particles are attracted to the tube wall by the magnetic field. Lin - cells remain in suspension and can be recovered by simply pouring off the liquid into a new tube. The Lin + cells remain in the original tube. (B) Mouse and human HSPCs can be enriched using columns for magnetic cell separation. Cells are incubated with cell enrichment cocktail followed by incubation with magnetic beads. Left. Enrichment of mouse Lin - cells is performed with the LS Miltenyi columns; enrichment of human CD34 + cells is performed with the MS Miltenyi columns. The column is mounted into the MidiMACS separator on the MACS MultiStand and prepared by adding column buffer. Center. The cell suspension is added to the top of the column. Right. The cell suspension is left to flow through the column via gravity flow. Cells bound by the magnetic particles remain bound to the column in the magnetic field, whereas unbound cells are recovered into a clean tube. (C) Mouse LT-HSCs can be purified from the enriched population of Lin - cells using FACS and a 5-flurochrome strategy based on the SLAM markers. Left. Detection of Lin - cells. Center. LSK cells within the Lin- population are recognized by being c-Kit+ Sca+. Right. LT-HSCs within the LSK population are recognized by being CD150+ CD48-

Article Snippet: APC-Cy7 anti-mouse CD48 (clone HM48-1); diluted 1:40∗ , Thermo Fisher Scientific , Cat# 47-0481-82; RRID: AB_2573962.

Techniques: Incubation, Suspension, Magnetic Cell Separation, Magnetic Beads, Purification

Journal: STAR Protocols

Article Title: Isolation of human and murine hematopoietic stem cells for DNA damage and DNA repair assays

doi: 10.1016/j.xpro.2021.100846

Figure Lengend Snippet: Generation of single-color controls for sorting of mouse LT-HSC

Article Snippet: APC-Cy7 anti-mouse CD48 (clone HM48-1); diluted 1:40∗ , Thermo Fisher Scientific , Cat# 47-0481-82; RRID: AB_2573962.

Techniques: Control

Journal: STAR Protocols

Article Title: Isolation of human and murine hematopoietic stem cells for DNA damage and DNA repair assays

doi: 10.1016/j.xpro.2021.100846

Figure Lengend Snippet:

Article Snippet: APC-Cy7 anti-mouse CD48 (clone HM48-1); diluted 1:40∗ , Thermo Fisher Scientific , Cat# 47-0481-82; RRID: AB_2573962.

Techniques: Recombinant, Electron Microscopy, Software, Blocking Assay, Single Cell Gel Electrophoresis