Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Mass Cytometry Assessment of Cell Phenotypes and Signaling States in Human Whole Blood

doi: 10.1007/978-1-0716-2553-8_10

Figure Lengend Snippet: Whole blood phosphoflow panel 1

Article Snippet: 142 Nd cCasp3 D3E9 Fluidigm 3142004A 143 Nd CD19 HIB19 Biolegend 302202 144 Nd pPLCg2 [Y759] K86-689.37 Fluidigm 3144015A 145 Nd CD4 RPA-T4 Fluidigm 3145001B 146 Nd IgD IA6-2 Fluidigm 3146005B 147 Sm CD20 2H7 Fluidigm 3147001B 148 Nd IgA Polyclonal Fluidigm 3148007B 149 Sm CD25 2A3 Fluidigm 3149010B 150 Nd pStat5 [Y694] 47 Fluidigm 3150005A 151 Eu CD123 6H6 Fluidigm 3151001B 153 Eu pStat1 [Y701] 4a Fluidigm 3153005A 154 Sm CD45 HI30 Fluidigm 3154001B 155Gd CD27 L128 Fluidigm 3155001B 156 Gd p38 [T180/Y182] D3F9 Fluidigm 3156002A 157 Gd CD24 ML-5 Biolegend 311102 158 Gd pStat3 [Y705] 4/P-Stat3 Fluidigm 3158005A 159Tb CD11c Bu15 Fluidigm 3159001B 160Gd CD14 M5E2 Fluidigm 3160001B 161 Dy CD141(BDCA-3) AD5-14H12 Miltenyi 130-090-694 162 Dy CD66b 80H3 Fluidigm 3162023B 163 Dy CD56 NCAM16.2 Fluidigm 3163007B 164 Dy IkBa L35A5 Fluidigm 3164004A 165 Ho pCREB [S133] 87G3 Fluidigm 3165009A 166 Er CD16 B73.1 Biolegend 360702 167 Er CD38 HIT2 Fluidigm 3167001B 168 Er CD8 SK1 Fluidigm 3168002B 169 Tm CD45RA HI100 Fluidigm 3169008B 170 Er CD3 UCHT1 Fluidigm 3170001B 171 Yb pERK1/2 [T202/Y204] D13.14.4E Fluidigm 3171010A 172 Yb Anti-Ki-67 B56 Fluidigm 3172024B 174 Yb HLA-DR L243 Fluidigm 3174001B 175Lu CD7 CD7-6B7 Biolegend 343102 176 Yb CD127/IL-7Ra P48-48 Novus Bio MAB306-100 209Bi CD11b/Mac-1 ICRF44 Fluidigm 3209003B Open in a separate window 1 Open channels are not shown but include Pd channels, Cd channel, Pt channels, 89Y,152Sm and 173Yb Whole blood phosphoflow panel1.

Techniques:

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Flow cytometric gating strategy adopted to identify CD33+/CD34+ precursor cells. A representative JMML patient is showed. Mononuclear cells were initially gated to exclude debris and residual granulocytes by physical parameters ( a ); all myeloid cells were selected by their reactivity to anti-CD33 antibody ( b ); myeloid precursors were then identified as CD33+/CD34+ double positive cells ( c ). CD33+/CD34+ cells were further checked for their negativity to anti-CD14 antibody ( d ) and low expression of CD45 ( e ), as features of myeloid precursor cells. p-STAT5 response was then measured on these selected cells by dual SSC/STAT5 cytogram ( f ). In panel ( e ), only CD33+/CD34+/CD14− gated cells are shown. In panel ( f ), only CD33+/CD34+/CD14−/CD45low gated cells are shown.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques: Expressing

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Training set samples assessed the best threshold. The p-STAT5 responses were measured at each GM-CSF concentration in the training set of samples (11 JMMLs and 23 controls). The best dose to distinguish JMML from non-JMML samples was identified at 0.1 ng/ml of GM-CSF ( P <0.0001). p-STAT5-positive cells (%) were quantified by scaling the maximum % of p-STAT5+ cells at 100 and the unstimulated p-STAT5+ cells to 0.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques: Concentration Assay

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Representative flow cytometric contour plots of p-STAT5 response in CD33+/CD34+ cells. Dual SSC/STAT5 cytograms from a JMML (upper panels) and a control (lower panels) are shown. Contour plots are referred to CD33+/CD34+ cells identified by gating strategy described in . For each dose of GM-CSF, the raw percentage of responding p-STAT5-positive cells is shown. Response to stimulation at each GM-CSF dose was then quantified by scaling the maximum percentage of p-STAT5+ cells at 100 and the unstimulated p-STAT5+ cells to 0. According to this criteria, calculated p-STAT5 responses are indicated in parenthesis for each stimulation dose.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Comparison of p-STAT5-positive cells (%) induced by 0.1 ng/ml of GM-CSF in the validation series comprising 11 JMML samples (central box plot), 24 controls (left box plot) and 7 samples from patients with other diseases mimicking JMML at presentation (right box plot). The discriminating threshold (17.17% as assessed in the training set) is indicated. The bold line inside each box plot indicates the median level, while the upper and lower lines indicate the maximum and minimum observed values, respectively. There are no outliers.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Comparison of p-STAT5-positive cells (%) induced by 0.1 ng/ml of GM-CSF according to the different cell source. In all, 17 JMML BM samples (left box plot), 5 JMML PB samples (middle left box plot), 12 BM samples and 2 PB samples (middle right and right box plot, respectively) from patients with other diseases mimicking JMML are shown. The discriminating threshold (17.17% as assessed in the training set) is indicated. The bold line inside each box plot indicates the median level, while the upper and lower lines indicate the maximum and minimum observed values, respectively. There are no outliers.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: CytoJournal

Article Title: Association of immunosuppressive CD45 + CD33 + CD14 − CD10 − HLA-DR −/low neutrophils with poor prognosis in patients with lymphoma and their expansion and activation through STAT3/arginase-1 pathway in vitro

doi: 10.25259/Cytojournal_165_2024

Figure Lengend Snippet: List of primer sequences used for RT-PCR analysis.

Article Snippet: After blocking with 5% bovine serum albumin (Gibco, Waltham, MA, USA), the membranes were incubated overnight at 4°C with the following primary antibodies: b-actin (1 μg/mL, Cat.No AF5003, Beyotime, Shanghai, China), STAT1 (2 μg/mL, Cat.No YT4439), STAT3 (2 μg/mL, Cat.No YT4443), STAT5 (2 μg/mL, Cat.No YT4453), phospho-STAT1 (p-STAT1, 2 μg/mL, Cat.No YP0249), phospho-STAT3 (p-STAT3, 2 μg/mL, Cat.No YP0250), and phospho-STAT5 (p-STAT5, 2 μg/mL, Cat.No YP0254) (all antibodies from ImmunoWay Biotechnology, USA, except b-actin).

Techniques:

Journal: CytoJournal

Article Title: Association of immunosuppressive CD45 + CD33 + CD14 − CD10 − HLA-DR −/low neutrophils with poor prognosis in patients with lymphoma and their expansion and activation through STAT3/arginase-1 pathway in vitro

doi: 10.25259/Cytojournal_165_2024

Figure Lengend Snippet: Increased activity of STAT3, which is required for T-cell suppression. (a 1 ) Neutrophils before and after induction were tested for STAT1, STAT3, STAT5, p-STAT1, p-STAT3, and p-STAT5 expression by WB. (a 2 ) Grayscale analysis of WB results by image J software; the result was expressed as the gray value of the target band/the gray value of the reference protein. Increased expression levels of STAT3 and p-STAT3 proteins were found in neutrophils AI compared with those before induction. (b) Changes of mRNA expression of STAT1, STAT3, and STAT5 in neutrophils before and after induction. The mRNA level of STAT3 in neutrophils AI was significantly higher than that in neutrophils before induction. (c 1 and c 2 ) Induced neutrophils with or without ruxolitinib were cocultured or indirectly cocultured with autologous CD3 + T cells from the same donors at 2:1 ratio for 48 h; T-cell proliferation was evaluated by CFSE labeling, and unstimulated T cells were used as a negative control. The samples were analyzed by flow cytometry. Ruxolitinib addition almost completely abolished the ability of CD10 − neutrophils to suppress T-cell proliferation. (c 3 ) Representative flow cytometry data of T-cell proliferation from one individual in coculture and indirect coculture systems. The red area represented the proliferating T-cell fraction. (d 1 and d 2 ) Induced neutrophils with or without ruxolitinib were cocultured or indirectly cocultured with autologous CD3 + T cells from the same donors at 2:1 ratio for 48 h, and apoptotic T cells were assessed by flow cytometry. CD3 + T cells cultured alone were used as a negative control. Ruxolitinib addition almost completely prevented T-cell apoptosis. (d 3 ) Representative flow cytometry data of CD3 + T-cell apoptosis experiment from one individual in the coculture or indirect coculture system. CD3 + T cells without PI or annexin V were used as a blank control. Apoptotic T cells were marked as CD3 + /annexin + /PI + . (e 1 ) WB analysis of the changes in STAT3 and p-STAT3 expression before and after ruxolitinib addition in the induced neutrophil population and (e 2 ) grayscale analysis of WB results by imageJ software. Ruxolitinib inhibited the expression of STAT3 and p-STAT3 proteins. (f) Extracellular production of Arg-1 protein was evaluated by ELISA at 48 h before and after the addition of ruxolitinib in neutrophils AI . Significantly decreased expression of extracellular Arg-1 in neutrophils AI treated with ruxolitinib. (g) Changes in intracellular Arg-1 fluorescence intensity were detected by flow cytometry at 48 h before and after ruxolitinib addition in neutrophils AI . Significantly decreased expression of intracellular Arg-1 in neutrophils AI treated with ruxolitinib, mainly in the CD10 − neutrophil subset. (h) Arg-1 activity was determined at 48 h before and after ruxolitinib addition in neutrophils AI . A significant decrease in Arg-1 activity was observed in the cells treated with ruxolitinib. (i) mRNA expression of Arg-1 was detected by RT-PCR in neutrophils AI at 48 h before and after ruxolitinib addition. A significant decrease was observed in the mRNA expression of Arg-1 in the cells treated with ruxolitinib. (j) mRNA expression of STAT3 was detected by RT-PCR in neutrophils at 48 h before and after ruxolitinib addition. The expression of STAT3 gene in CD10 − neutrophil subpopulation significantly decreased. Each experiment was repeated 3 times. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001, ✶ ✶ ✶ ✶ P < 0.0001, ns: P > 0.05. Ctrl: Neutrophils before induction, neutrophils AI : Neutrophils after induction, WB: Western blot, CFSE: Carboxyfluorescein succinimidyl ester, CD10 − neutrophils: CD45 + CD14 − CD33 + HLA-DR − CD10 − cells, CD10 + neutrophils: CD45 + CD14 − CD33 + HLA-DR − CD10 + cells.

Article Snippet: After blocking with 5% bovine serum albumin (Gibco, Waltham, MA, USA), the membranes were incubated overnight at 4°C with the following primary antibodies: b-actin (1 μg/mL, Cat.No AF5003, Beyotime, Shanghai, China), STAT1 (2 μg/mL, Cat.No YT4439), STAT3 (2 μg/mL, Cat.No YT4443), STAT5 (2 μg/mL, Cat.No YT4453), phospho-STAT1 (p-STAT1, 2 μg/mL, Cat.No YP0249), phospho-STAT3 (p-STAT3, 2 μg/mL, Cat.No YP0250), and phospho-STAT5 (p-STAT5, 2 μg/mL, Cat.No YP0254) (all antibodies from ImmunoWay Biotechnology, USA, except b-actin).

Techniques: Activity Assay, Expressing, Software, Labeling, Negative Control, Flow Cytometry, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of IGF-IR tyrosine kinase induces apoptosis and cell cycle arrest in imatinib-resistant chronic myeloid leukaemia cells

doi: 10.1111/j.1582-4934.2009.00795.x

Figure Lengend Snippet: Effects of inhibition of IGF-IR on IGF-IR, BCR-ABL and downstream target proteins in CML cell lines. (A) PPP induces concentration-dependent decrease in IGF-IR tyrosine kinase activity in K562 and KBM-5 cell lines. In contrast, PPP fails to cause similar effect in BCR-ABL tyrosine kinase activity. The results represent the means ± S.D. of three consistent experiments. *: P < 0.01 and †: P < 0.001 compared with control untreated cells. (B) Western blotting and co-immunoprecipitation studies confirm that PPP decreases the tyrosine phosphorylation of IGF-IR in a concentration-dependent fashion (results shown are representative and were obtained from the KBM-5 cell line). The basal levels of IGF-IR did not change after treatment with PPP. The phosphorylation level of BCR-ABL remains unchanged after treatment with PPP. The decrease in pIGF-IR is associated with down-regulation of pAkt and pSTAT5, two oncogenic proteins in CML. Changes are not seen in Akt and STAT5. Also, PPP induces changes consistent with apoptotic cell death including down-regulation of Bcl-2, Bcl-X L and caspase-3. Moreover, treatment with PPP induces up-regulation of cyclin B1 and down-regulation of cyclin E and pCdc2, whereas the levels of Cdc2 and p16 remain unchanged. Overall, the changes in the cell cycle regulatory proteins are consistent with G2/M-phase cell cycle arrest. β-Actin shows equal loading of the proteins. (C) IGF-IR siRNA decreases IGF-IR levels in the KBM-5 cell line and this decrease is associated with down-regulation of pAkt and pSTAT5. Basal levels of these two proteins remain unchanged. IGF-IR siRNA did not induce alterations in BCR-ABL or pBCR-ABL protein. β-Actin confirms equal loading of the proteins.

Article Snippet: Antibodies obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) included Bcl-2 (catalogue number: sc-7382), cyclin B1 (sc-7393), cyclin E (sc-198), Cdc2 (sc-52316), pCdc2 (Thr14/Tyr15; sc-12340-R) and p16 (sc-56330); from Cell Signaling Technology (Danvers, MA, USA) were pIGF-IR (Tyr1131; 3021), pBCR-ABL (p-c-Abl; Tyr412; 2865), Akt (9272) and pAkt (Ser473; 587F11); from Zymed Laboratories (South San Francisco, CA, USA) were IGF-IRβ (39–6700) and Bcl-X L (18–0217); from Calbiochem (Gibbstown, NJ, USA) was BCR-ABL (c-Abl; OP19); from R&D Systems (Minneapolis, MN, USA) was STAT5 (MAB2174); from GeneTex Incorporation (San Antonio, TX, USA) was pSTAT5 (Tyr694; GTX52364) and from Sigma (St. Louis, MO, USA) was β-Actin (A-2228).

Techniques: Inhibition, Concentration Assay, Activity Assay, Western Blot, Immunoprecipitation