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13 protocols using apc conjugated anti cd44

1

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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2

Stem Cell Marker Profiling by Flow Cytometry

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Allophycocyanin (APC)-conjugated anti-CD44 and phycoerythrin (PE)-conjugated anti-CD24 monoclonal antibodies (BD Biosciences, USA) were used to stain stem cell markers. After digestion, the cells were washed with PBS. Subsequently, 1 × 106 cells were resuspended in 100 μl PBS before adding APC-CD44 and its isotype control APC-IgG or PE-CD24 and its isotype control PE-IgG. The cells were incubated at 4 °C for 40 min, washed in PBS and then analyzed using a flow cytometer. Gating parameters were defined according to the cells labeled with the relevant isotype control (APC-IgG and PE-IgG).
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3

Characterization of Cancer Stem Cells

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For the analysis of CD44 positive and CD133 positive cells, cells were stained with PE-conjugated anti-CD133 (Miltenyi Biotec) and APC-conjugated anti-CD44 (BD) antibody.
For apoptosis analysis, cells were stained with Annexin V-FITC Apoptosis Kit (K101, Biovision, Milpitas, CA, USA) according to the manufacturer’s instructions.
After the indicated labeling, cells were analyzed with MoFlo Astrios (Beckman-Coulter, CA, USA).
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4

Immunoblotting and Flow Cytometry Antibodies

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Antibodies for immunoblotting included anti-EGFR_pY1068 (#D7A5), anti-total EGFR (#D38B1), anti-ERK1/2_pT202/Y204 (#D13.14.4E), anti-total ERK1/2 (#137F5), and anti-MEK1/2 (#D1A5), all from Cell Signaling Technology. Antibodies for flow cytometry, PE-conjugated anti-CD24 (#ML5) and APC-conjugated anti-CD44 (#IM7), were from BD Biosciences and Affymetrix eBioscience, respectively.
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5

Thymocyte Apoptosis Profiling by TUNEL and Annexin V

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Single cell suspension of thymocytes was prepared from the mice and stained with biotinylated antibodies against lineage markers B220, CD4, CD8, Gr-1, Mac-1, NK1.1, and γδTCR, followed by PerCPCy5.5-conjugated streptavidin, APC-conjugated anti-CD44, and PECy7-conjugated anti-CD25 (all from BD Biosciences or eBioscience). Then TUNEL assay was performed using In Situ Cell Death Detection Kit, TMR red (Roche Life Science) following the manufacturer’s instructions. For Annexin V staining, thymocytes were stained with PE-conjugated antibodies against the same lineage markers, APCCy7-conjugated anti-CD44, PECy7-conjugated anti-CD25, and APC-conjugated Annexin V. DAPI was added to the samples just before the cells were examined on LSR II Flow Cytometer. Data was analyzed using FACSDiva software (BD Biosciences).
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6

Isolation of CD44+, CD133+ Cells

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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).
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7

Comprehensive Stemness and EMT Marker Profiling

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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).
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8

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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9

Quantifying CD133 and CD44 in Cell Populations

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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)].
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10

ZIKV E Antigen-Stimulated Splenocyte Response

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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).
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