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Pe conjugated cd24

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PE-conjugated CD24 is a fluorochrome-labeled antibody that binds to the CD24 surface antigen. CD24 is a glycosylphosphatidylinositol (GPI)-anchored sialoglycoprotein that is expressed on a variety of cell types, including hematopoietic cells and some epithelial cells. The PE (phycoerythrin) fluorescent label allows for the detection and analysis of CD24-expressing cells using flow cytometry or other fluorescence-based applications.

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6 protocols using pe conjugated cd24

1

Characterizing Stem Cell Markers

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To access the levels of Aldefluor, CD24, CD44 and CD133 positive populations, the cells were stained with Aldefluor kit (Stem Cell Technologies, Vancouver, Canada, Cat. 01700, dilution 1/40), PE conjugated CD24 (BD Bioscience, San. Jose, CA, USA, Cat. 555428, dilution 1/40), APC conjugated CD44 (BD Bioscience, Cat. 559942, dilution 1/40) and PE conjugated CD133 (MACS; Miltenyi Biotech, Sunnyvale, CA, USA, 130-080-081, dilution 1/40), respectively. Flow cytometry data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). The flow cytometry gates were established by staining with an isotype antibody or secondary antibody.
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2

Breg Cell Enumeration by Flow Cytometry

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Evaluation of Breg cells frequencies by flow cytometry, circulating Breg cells were detected using FITC-conjugated-CD38, PE-conjugated-CD24 (BD Bioscience, USA), and PerCP-conjugated CD19 (BD Bioscience, USA).100 μl of blood sample was incubated with 10 μl of CD24, CD38, and CD19 for 20 minutes at 4 C in the dark. Following incubation, red blood cell lysis, washing, and analysis by FACS Calibur flow cytometry with Cell Quest software (Becton Dickinson Biosciences, USA) was done. An isotype-matched negative control was used for each sample. A Forward and side scatter histogram was used to define the lymphocytes. Then CD19+ B cells were gated. Then the expression of CD38 and CD24 on the CD19+B cells was revealed. Regulatory B cells were specified as CD19+CD24highCD38high cells (Figure 1).
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3

Stemness Marker Analysis of PANC-1 Cells

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Adherent PANC-1 cells (80–90% confluent) were collected and centrifuged. The concentration of cells was adjusted to 1 × 106 cells/ml. For analysis of stemness markers, cells (1 × 106) were labeled with PE-conjugated CD24 (BD Pharmingen™, cat. no. 555428; BD Biosciences, East Rutherford, N.J.), and APC-conjugated anti-CD44 (BD Pharmingen™, cat. No. 559942; BD Biosciences.) and incubated at 4 °C 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).
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4

Cell Surface Marker Profiling by Flow Cytometry

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Cell labeling was carried out by staining with antibodies in buffer composed of PBS supplemented with 1% BSA. Cells were first suspended and placed in a blocking solution on ice at a concentration of 1 × 105 cells in a total volume of 0.5 mL for 30 min and then stained with conjugated antibodies for 45 min on ice in the dark. After centrifugation at 250× g for 5 min at 4 °C, cells were washed with cold staining buffer before being subjected to flow cytometry. The antibodies used in this study were PE-conjugated CD 24 (BD Bioscience, Franklin Lakes, NJ, USA) and FITC-conjugated CD29 (Biolegend, San Diego, CA, USA). Proper isotype controls were used for each cell labeling experiment. The main cellular population was gated on FSC/SSC, then doublets were removed and the remaining cells were analyzed for percentages of cells showing CD29+/CD24+, CD29−/CD24+, CD29+/CD24−, or CD29−/CD24−. Flow cytometry was performed on a Guava 6HT-2L flow cytometer (Luminex, Austin, TX, USA) and data analyzed using FlowJo, LLC software (Mac Version 10.8.0 Ashland, OR, USA).
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5

Breast Cancer Cell Immunophenotyping

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We followed a previously described method [39 (link)]. A total of 1 × 106 cells were incubated with FITC-conjugated CD44 and PE-conjugated CD24 (BD) and incubated at 4 °C for 20 min. The breast cancer cells were washed twice with 1XPBS and then analyzed by using a flow cytometer (Accuri C6, BD).
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6

Stemness and Apoptosis Analysis of Pancreatic Cancer Cells

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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.
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