Pe cy5 anti mouse ly 6 a e

The following monoclonal antibodies were used to perform staining of Lin⁻/Sca-1⁺/c-Kit⁺ (SKL) cells and Lin⁻/Sca-1⁺/CD45⁺ (hematopoietic stem cells [HSCs]): FITC–anti-CD117 (also known as c-Kit, clone 2B8; BioLegend, San Diego, CA, USA) and PE–Cy5–anti-mouse Ly-6 A/E (also known as Sca-1, clone D7; eBioscience, San Diego, CA, USA). All anti-mouse lineage markers, (Lin) anti-CD45R/B220 (clone RA3-6B2), anti-Ter-119 (clone TER-119), anti-CD11b (clone M1/70), anti-T cell receptor β (clone H57–597), anti-Gr-1 (clone RB6-8C5), anti-TCRγδ (clone GL3), and anti-CD45 (clone 30-F11), were purchased from BD Biosciences and conjugated with PE as described. Staining was performed in RPMI 1640 medium containing 2 % FBS. All monoclonal antibodies (mAbs) were added at saturating concentrations, and the cells were incubated for 30 min on ice, washed twice, and analyzed with an LSR II flow cytometer (BD Biosciences).

For the staining of Lin^–/Sca-1⁺/c-Kit⁺ (SKL) cells, the following monoclonal antibodies were used: FITC–anti-CD117 (also known as c-Kit, clone 2B8; BioLegend, San Diego, CA, USA), PE–Cy5–anti-mouse Ly-6 A/E (also known as Sca-1, clone D7; eBioscience, San Diego, CA, USA), and anti-mouse lineage-marker antibodies, including anti-CD45R (also known as B220, clone RA3-6B2), anti-Ter-119 (clone TER-119), anti-CD11b (clone M1/70), anti-T cell receptor β (clone H57-597), anti-Gr-1 (clone RB6-8C5), and anti-TCRγδ (clone GL3) conjugated with PE (BD Biosciences, San Jose, CA, USA). Staining was performed in RPMI-1640 medium containing 2% FBS. All monoclonal antibodies were added at saturating concentrations, and the cells were incubated for 30 min on ice, washed twice, and analyzed using an LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) [4 (link), 7 (link), 26 (link)].

The following monoclonal antibodies were used to perform staining of Lin⁻/Sca-1⁺/c-Kit⁺ (SKL) cells and Lin⁻/Sca-1⁺/CD45⁺ hematopoietic stem cells (HSCs): FITC–anti-CD117 (also known as c-Kit, clone 2B8; BioLegend, San Diego, CA, USA) and PE–Cy5–anti-mouse Ly-6 A/E (also known as Sca-1, clone D7; eBioscience, San Diego, CA, USA). All anti-mouse lineage marker [5 (link)] antibodies, including anti-CD45R/B220 (clone RA3-6B2), anti-Ter-119 (clone TER-119), anti-CD11b (clone M1/70), anti-T cell receptor β (clone H57-597), anti-Gr-1 (clone RB6-8C5), anti-TCRγδ (clone GL3), and anti-CD45 (clone 30-F11), were purchased from BD Biosciences and conjugated with PE as described [35 (link), 44 (link)]. Staining was performed in RPMI 1640 medium containing 2% FBS. All monoclonal antibodies (mAbs) were added at saturating concentrations, and the cells were incubated for 30 min on ice, washed twice, and analyzed with an LSR II flow cytometer (BD Biosciences).

For staining of Lin⁻/Sca-1⁺/c-Kit⁺ (SKL cells), Sca-1⁺/Lin⁻/CD45⁻ (VSELs), Lin⁻/CD45⁻/CD31⁺ (EPCs), and Lin⁻/CD45⁻/CD31⁻/CD90⁺ (MSCs), the following monoclonal antibodies were used: FITC–anti-CD117 (also known as c-Kit, clone 2B8; BioLegend, San Diego, CA, USA) and PE–Cy5–anti-mouse Ly-6 A/E (also known as Sca-1, clone D7; eBioscience, San Diego, CA, USA). All anti-mouse lineage marker antibodies, including anti-CD45R (also known as B220, clone RA3-6B2), anti-Ter-119 (clone TER-119), anti-CD11b (clone M1/70), anti-T cell receptor β (clone H57–597), anti-Gr-1 (clone RB6-8C5), anti-TCRγδ (clone GL3), and anti-CD45 (clone 30-F11), conjugated with PE; anti-CD31 (clone MEC 13.3), conjugated with APC; and anti-CD90.2 (clone 30-H12), conjugated with BV510, were purchased from BD Biosciences. Staining was performed in RPMI-1640 medium containing 2% FBS. All monoclonal antibodies were added at saturating concentrations, and the cells were incubated for 30 min on ice, washed twice, and analyzed with an LSR II flow cytometer (BD Biosciences) [21 (link)].