Anti cd44 apc

Expression of human NSCLC stem cell marker CD133 and CD44 was analyzed by flow cytometric study in A549 cells and spheroids by using anti-CD133-FITC and anti-CD44-APC antibodies (BD Biosciences). CD133⁺ and CD44⁺ CSCs were flow-cytometrically gated from spheroids on the basis of the cell surface phenotype. Mean fluorescence intensities of Oct-4-PerCP-Cy5.5, Nanog-PE, Sox-2- Alexa Fluor-647, Mrp1-FITC, Aldh1-FITC (BD Biosciences) were quantified^{22 (link)}. Mean fluorescence intensities of Akt, p-Akt, Cxcr-4, Mdr1, Integrin-α2, Integrin-α5, Integrin-β1, p-Fak (Santa Cruz Biotechnology, Inc.) were determined with respective primary antibodies conjugated with PE as previously described^{13 (link)}.

Cancer cells dissociated from the transplanted tumor tissues or from culture plates were counted and re-suspended in 100 μl of HBSS (Gibco, Langley, OK, USA) containing 2% heat-inactivated fetal bovine serum (FACS buffer) and 10⁵ cells. Five microliters of mouse IgG solution (1 mg/ml) was added and incubated on ice for 10 min. According to the manufacturer’s recommendation, appropriate antibodies were added and incubated for 30 min on ice. The cells were then washed twice with FACS buffer and re-suspended in 0.2 ml of FACS buffer that contained 7-aminoactinomycin D (7-AAD, eBioscience, San Diego, CA, 1 μg/ml, final concentration) to exclude dead cells. Antibodies used were anti-CD44 (APC) and anti-CD24 (PE), which were purchased from BD Pharmingen. ALDH activity was examined with the ALDEFLUOR kit (Stem cell Technologies, Vancouver, Canada). Cell apoptosis was assessed using an Annexin V-PE-Cy7 Apoptosis Detection Kit (eBiosciences, Burlington, ON, Canada) for Beckman Cyan-ADP 9 flow cytometer or V450 Annexin V (BD) for BD LSR Fortessa. Flow cytometric data were analyzed with Kaluza software (Beckman Coulter, USA) or FlowJo (FlowJo LLC, Ashland, Oregon, USA).

To detect cell surface markers, the cells were trypsinized, blocked in ice-cold PBS containing bovine serum albumin (BSA, 2%) and 0.1% NaN₃, then incubated at 4°C with an anti-CD133/2 (293C3)-APC (Miltenyi Biotec), anti-CD44-APC or anti-CD24-PE (BD Pharmingen) antibody for 30 mins. ROS detection was done by DCFDA (Sigma) labeling followed by detection of the oxidized DCF signal. Cellular fluorescence was measured using 50,000 cells on an Accuri C6 Flow Cytometer (BD Biosciences). FACS was performed using an AriaSORT cell sorter (BD Biosciences). Data were processed using the FlowJo software package (Tree Star Inc.).

Single-cell suspensions generated in PBS supplemented with 1% FBS were incubated with anti-EpCAM-FITC (1.20, Genetex, GTX30708), and anti-CD44-APC (1.20, BD Pharmingen, 559250) antibodies for 30 min on ice and analyzed on a FACSAria III Cell Sorter (BD Biosciences). CD44^hiEpCAM^hiand CD44^hiEpCAM^lo HCT116 and SW480 cells were sorted and cultured in humidified atmosphere at 37°C with 5% CO₂ for 3–5 days before collecting RNA or protein, as previously described (Sacchetti et al., 2021 (link)). The subpopulation of cells mapping in between the CD44^hiEpCAM^hi and CD44^hiEpCAM^lo gates was labelled as intermediate and was further not employed for analysis.

Blood was sampled from the retro-orbital sinus of 15 mice once per month for 8 time points (total of 120 samples). Mononuclear cells from the peripheral blood was isolated by density gradient centrifugation using Ficoll (Ficoll PaqueTM plus, GE Health Care), Single cell suspensions were prepared from thymus and spleen that were removed from each mouse at the end of the experiment. For cell sorting, cells were stained with the following fluorescently labeled monoclonal antibodies: anti-CD4 Pacific Blue (BD), anti-CD25 PE (eBioscience), anti-CD44 APC (BD) and anti-CD62L PE-Cy7 (eBioscience) and viability using the Fixable Viability stain 450 (BD Horizon). Cell sorting was performed using FACS ARIA III sorter. CD4+ D44loCD62Lhi were sorted as naive T cells. After sorting, cells were pelleted and resuspended with 300μl of RNA protect cell reagent (Qiagen). Cells were stored at minus 80°C until RNA extraction. RNA was purified from RNAprotect-stabilized cells using the RNeasy Plus Mini Kit. After RNA extraction, samples were run on TapeStation to estimate quality.