Cd44 pe

Mouse spleens were homogenized, and single-cell suspensions were prepared and counted after lysis with RBC lysis buffer (BioLegend). For macrophage staining, 1 × 10⁶ cells were incubated with FITC-F4/80 (Biolegend) and APC-Ly6c (Biolegend) antibodies at room temperature for 30 min. The cells were rinsed with 1 ml PBS and resuspended with 200 μl PBS for detection. For the detection of Tcm (central memory T cell) and Tem (effector memory T cell), 1 × 10⁶ cells were incubated with FITC-CD62L (Biolegend), PE-CD44 (Biolegend), Percp-cy5.5-CD4 (Biolegend), and APC-CD8 (Biolegend) antibodies for 30 min. The cells were then subjected to 1 ml PBS washing, followed by suspending with 200 μl PBS for cytometry detection. For intracellular staining, 2 × 10⁶ cells were stimulated with PMA plus ionomycin (Invitrogen) for 5 hr. After incubation with FITC-CD4 (Biolegend) and PE-CD8 (Biolegend) antibodies at room temperature for 30 min, the cells were then incubated with Fix/Perm according to kit instructions and APC-IFN-γ (Biolegend) antibodies for 30 min. Then the cells were rinsed with 1 ml PBS and resuspended with 200 μl PBS for detection.

ALDEFLUOR assay was performed following the manufacturer's protocol (STEMCELL). For staining of fluorescence-conjugated antibodies, namely FITC-CD24 (1:40), PE-CD44(1:20) and APC-NOTCH4 (1:40, BioLegend, CA, USA), against certain cell surface markers, cells suspended in antibody diluent was incubated on ice for 30 min and then washed twice with cold PBS. When combined staining of ALDEFLUOR assay and NOTCH4 was performed, ALDEFLUOR assay was carried out first. For cell cycle analysis, cells were fixed in 70% alcohol at 4 °C for at least 4 h, followed by staining with propidium iodide (Sigma) in the presence of 1% RNase A (Takara) at 37 °C for 30 min. For apoptosis analysis, cells were stained with propidium iodide (Sigma) and APC-Annexin V (BD), according to the manufacturer's recommendations. Analysis of the samples were completed on Moflo Astrios or CytoFlex (Beckman Coulter), equipped with a 405/448 nm channel for DAPI to delineate dead cells, a 488/513 nm channel for ALDEFLUOR or FITC, a 561/579 nm channel for PE, and a 640/671 channel for APC detection.

The cells were digested and suspended in serum-free medium, and the cellular concentration was adjusted to 1 × 10⁷ cells/mL. To the cell suspension was added the appropriate amount of direct labeled flow antibody FITC-CD133 (Biolegend), PE-CD44 (Biolegend), and it was incubated for 30 min at 4 °C. Labeled cells were washed with serum-free medium twice and samples resuspended with 300–500 μL/tube serum-free medium and were detected with BD LSRFortessa. Data analysis was performed in Flowjo.6.1.

AMD1 expression/knockdown lentiviruses and their corresponding negative controls without fluorescence labeling were used for stable cell lines establishment. After transfected, cells were incubated with the following antibodies: APC‐CD90 (BioLegend, 328113) and PE‐CD44 (BioLegend, 103007). The subsequent steps were performed as previously described.²⁸

After the mice were sacrificed, the tumors were collected and digested in dissociation buffer [mixture of deoxyribonuclease (50 μg mL^–1), hyaluronidase (100 μg mL^–1), and collagenase IV (2 mg mL^–1)] at 37 °C for 1 h with gentle shaking. The cell suspension was passed through a 70 μm cell strainer and dispersed in PBS buffer to form single-cell suspension. For T lymphocytes detection, the single-cell suspension was stained with antibodies against CD3, CD4 and CD8 to mark the helper (CD3⁺ and CD4⁺) and cytotoxic (CD3⁺ and CD8⁺) T cells. The tumor-cell suspension was stained with antibodies against CD11b, F4/80, CD86, and CD206 to analyze M1-like (CD11⁺ and CD86⁺) and M2-like (F4/80⁺ and CD206⁺) macrophages. The mice’s blood and spleen were also collected for lymphocyte extraction using lymphocyte isolation kit (Solarbio) according to the manufacturer’s manual. NK cells (CD3⁻, CD49b⁺), γδ T cells (CD3⁺, TCRβ⁻), and memory T cells (CD8⁺, CD44^high, CD62L^low) were analyzed using flow cytometer. The anti-mouse of CD3-PE (clone: 17A2), CD4-APC-cy7 (clone: GK1.5), CD8a-FITC (clone: 53-6.7), CD11b-PE (clone: M1/70), F4/80-APC (clone: BM8), CD86-PE-Cy7 (clone: GL-1), CD206-FITC (clone: C068C2), CD49b-APC (clone: HMα2), TCRβ-FITC (clone: H57-597), CD44-PE (clone: IM7), and CD62L-APC-Cy7 (clone: MEL-14) were purchased from Biolegend.