Apc conjugated anti cd44

As in flow cytometry, cells were detached in the same conditions. Samples were incubated with APC-conjugated anti CD44 (BD Bioscience), PE-conjugated anti-CD133 or a mix of ant-CD44/CD133 during 20 min at 4°C in darkness. Cells were washed. Samples were filtrated and collected in special cell cytometry sterile tubes. Cell sorting was performed to separate CD44+, CD133+ or CD44+/CD133+ (double positive) cells in a MoFlow Sorter. Later cells were seeded in different culture media types (DMEMF-12 with 5% (v/v) FBS, colony formation medium with ITS, or 3D culture media).

COMMD1 (NBP2-4633, Novusbio), ZEB1 (NBP2-13159, Novusbio), Sox2 (ab97959, Abcam), KLF4 (ab151733, Abcam), Oct-4 (2750, Cell Signaling), MYC (5605, Cell Signaling), NANOG (D73G4)(4903, Cell Signaling), K8/18 (20R-Cp004, Fitzgerald Industries), P53 (SC-126, Santa Cruz Biotechnology), Phospho-NFκB p65 (Ser536) (93H1)(3033, Cell Signaling), HIF1A(610959, BD), HIF2A(NB100-122, Novusbio), MCT4(sc-50329, scbt), CAV1(610407, BD), B-actin (A5441, Sigma), tubulin (T4026, Sigma), FITC-conjugated anti-CD24, PE-conjugated anti-CD24, APC-conjugated anti-CD44, APC-conjugated anti-PD-L1 (BD Biosciences).

The pancreatic cancer cells in the logarithmic growth stage were collected and centrifuged. The concentration of cells was adjusted to 1×10⁶/ml.
For analysis of stemness markers, cells (1×10⁶) were labeled with PE‐conjugated CD24 (BD PharMingen™, cat. no. 555428) and APC‐conjugated anti‐CD44 (BD PharMingen™, cat. no. 559942), incubated at 4℃ in the dark for 30 min. Cells were then centrifuged and resuspended in PBS at a concentration of 1×10⁸/ml, and subsequently sorted on a flow cytometer (BD Accuri C6 Plus; BD Biosciences).
For apoptosis and necrosis analysis, cells were cultured for 12 h prior to being treated with gem for a further 24 h. For flow cytometry analysis, cells were detached and labeled using an Annexin V‐PE/7‐AAD Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd.; cat. no. KGA1017) according to the manufacturer's protocol. Apoptotic and necrotic cell subsets were quantified using flow cytometry. A total of 2×10⁴ cells were analyzed per sample. Annexin V‐PE⁻/7‐AAD⁻ cells were considered as viable, Annexin V‐PE⁺/7‐AAD⁻ cells were considered as early apoptotic, and Annexin V‐PE⁺/7‐AAD⁺ cells were considered as late apoptotic and necrotic cell populations.

Single-cell suspensions were prepared and washed in PBS containing 1% BSA. Next, the cells were incubated with PE-conjugated anti-CD133 (566593, BD Pharmingen, San Diego, CA, USA) and APC-conjugated anti-CD44 (559942, BD Pharmingen) antibodies for 15 min at 4 °C. Labeled cells were washed again and suspended in PBS. Flow cytometry (BD Biotechnology) was used to detect the expression of CD133 and CD44. The apoptosis experiment was carried out following the protocol of our previous study [44 (link)].

A total of 5×10⁵ splenocytes (treated as in the splenocyte proliferation assay) along with purified ZIKV E antigen (10 μg/ml) were plated in 96-well plates. After culture for 72 h, the cells were collected in PBS containing 2% FBS and 0.1% NaN₃. Then, the cells were stained with 0.25 μg of FITC-conjugated anti-CD4, PE-conjugated anti-CD8, APC-conjugated anti-CD19, PE/Cy7-conjugated anti-CD69, APC-conjugated anti-CD44 or PerCP-Cy5.5-conjugated anti-CD62L (BD Biosciences, Franklin, CA, USA) antibodies for 30 min at 4°C. Data were acquired on a FACSCalibur flow cytometer (BD Biosciences, Franklin, CA, USA).