Anti-mouse rat antibodies (conjugated with FITC, PE, PE-Cyanine7, PE-CF594, PerCP, APC, Alexa Fluor® 488, and BV510 as indicated) used in this study were: CD4-PerCP (L3T4, BD Pharmingen™, United States), CD25-APC (IL-2 Receptor α chain, BD Pharmingen™, United States), FoxP3-PE-Cyanine7 (JM2, eBioscience, United States), CD45RA-PE (BD Pharmingen™, United States), IL-17-Alexa Fluor® 488 (BD Pharmingen™, United States), CD3e-PE-CF594 (CD3ε chain, BD Horizon™, United States), CD8a-BV510 (Ly-B, BD Horizon™, United States). In all experiments, a control antibody of the respective IgG isotype was included. Leukocyte Activation Cocktail (BD Pharmingen™, United States), RNeasy Micro Kit (QIAGEN, Germany), FastQuant RT Kit (TIANGEN, China), SuperReal PreMix Plus (TIANGEN, China), and the Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences, United States) were used according to the manufacturer’s instructions.
Cd45ra pe
Manufactured by BD
Sourced in United States
CD45RA-PE is a fluorochrome-labeled monoclonal antibody used for the identification and enumeration of CD45RA+ cells in flow cytometry applications. CD45RA is a specific isoform of the CD45 protein tyrosine phosphatase, which is expressed on the surface of certain types of T cells and B cells.
Automatically generated - may contain errors
Lab products found in correlation
Igd fitc, by BD (3 mentions)
Cd19 fitc, by BD (3 mentions)
Cd3 fitc, by Miltenyi Biotec (2 mentions)
Cd62l apc, by Miltenyi Biotec (2 mentions)
Facsverse, by Merck Group (2 mentions)
Slc29a2 polyclonal antibody, by Thermo Fisher Scientific (2 mentions)
Ifn γ pe cy7, by BD (2 mentions)
Cd8 apc h7, by BD (2 mentions)
Cd3 percp cy5, by BD (2 mentions)
Ccr7 fitc, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Cd38 pe, by BD (2 mentions)
Cd27 pe, by BD (2 mentions)
Tim 3 pe, by BD (2 mentions)
Cd45ro apc, by BD (2 mentions)
Cd8 fitc, by BD (2 mentions)
Cd3 fitc, by BD (2 mentions)
Cd56 pe, by BD (2 mentions)
Cd14 fitc, by BD (2 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (2 mentions)
22 protocols using cd45ra pe
1
Murine Immune Cell Immunophenotyping
2
PBMC Phenotyping and hENT2 Expression
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PBMC samples were seeded (1 × 104 cells/well) in complete medium with or without the indicated concentrations of compounds. After 72 h incubation, cells were washed with PBS and stained with CD45RA PE (BD-Biosciences), CD62L APC (Miltenyi Biotec), CD3 FITC (Miltenyi Biotec) at room temperature for 20 min. Data collection was done on FACSVerse and dead cells were excluded from the analysis, based on the use of PI (Sigma-Aldrich).
For the evaluation of hENT2 protein expression, cell lines or AML primary cells were incubated with a SLC29A2 polyclonal antibody (Thermo fisher Scientific) using a dilution of 1:25 for 20 min at room temperature after fixation and permeabilization. The secondary antibody (Cell signaling Technology USA, Danvers, MA, USA) was used with a dilution of 1:200 for 20 min. Results were expressed as ΔGmean, i.e., the net mean fluorescence intensity (MFI) difference between the cells stained with primary and secondary antibodies and cells stained with the secondary antibody only, in order to eliminate the background positivity. All data were processed with Kaluza software version 1.2 (Beckman Coulter).
For the evaluation of hENT2 protein expression, cell lines or AML primary cells were incubated with a SLC29A2 polyclonal antibody (Thermo fisher Scientific) using a dilution of 1:25 for 20 min at room temperature after fixation and permeabilization. The secondary antibody (Cell signaling Technology USA, Danvers, MA, USA) was used with a dilution of 1:200 for 20 min. Results were expressed as ΔGmean, i.e., the net mean fluorescence intensity (MFI) difference between the cells stained with primary and secondary antibodies and cells stained with the secondary antibody only, in order to eliminate the background positivity. All data were processed with Kaluza software version 1.2 (Beckman Coulter).
3
Comprehensive Immune Cell Profiling
4
Comprehensive Immune Cell Profiling
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The following conjugated antibodies were used for cell-surface staining: CD3-Pacific Blue (BD Pharmingen; Clone UCHT1; Cat. No. 558117; San Diego, CA, USA), CD8-APC H7 (BD Pharmingen; Clone SK1; Cat. No. 641400), CD45RA-PE (BD Pharmingen; Clone HI100; Cat. No. 555489), CCR7-PE-Cy7 (BD Pharmingen; Clone 3D12; Cat. No. 557648), CD28-PerCP-Cy5.5 (BD Biosciences; Clone L293; Cat. No. 337181; San Jose, CA, USA), CD27-Alexa Fluor 700 (BD Pharmingen; Clone M-T271; Cat. No. 560611), CD95-APC (BD Pharmingen; Clone DX2; Cat. No. 558814) and CD127-FITC (BD Pharmingen; Clone HIL-7R-M21; Cat. No. 560549). Conjugated antibodies for intracellular staining included the following: IFN-γ-FITC (BD Pharmingen; Clone 4S.B3; Cat. No. 554551), IL-2-PerCP-Cy5.5 (BD Pharmingen; Clone MQ1-17H12; Cat. No. 560708) and TNF-α-AlexaFluor 700 (BD Pharmingen; Clone MAb11; Cat. No. 557996). To exclude dead cells, the Fixable Aqua Dead Cell Stain viability marker was used (Invitrogen; Cat. No. L34957; Eugene, OR, USA).
5
Immunophenotyping of T Cell Subsets
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Routine assessment of differential blood counts was used to define the engraftment of neutrophil leukocytes and reconstitution of whole lymphocytes. Samples for immunophenotyping were taken on day 60, day 120 and day 180 following transplantation. Twenty healthy stem cell donors were analyzed to define normal ranges (controls).
CD4+ and CD8+ T cells were characterized according to the expression of CCR7 and CD45RA as naïve (CCR7+CD45RA+), central memory (CM, CCR7+CD45RA−), effector memory (EM, CCR7−CD45RA−), and terminally differentiated effector memory (TEMRA, CCR7− CD45RA+) T cells.17 (link) Staining was performed using the following antibodies as previously described:18 (link) CD45-V500, CD3-PerCP-Cy5.5, CD8-APCH7, CCR7-FITC, CD45RAPE (all BD Biosciences, San Jose, CA, USA) and CD4-eFluor450 (eBioscience, San Diego, CA, USA) (Online Supplementary Figure S1). Immunophenotyping was performed using a BD FACSCanto II (BD Bioscience, San Jose, CA, USA).
CD4+ and CD8+ T cells were characterized according to the expression of CCR7 and CD45RA as naïve (CCR7+CD45RA+), central memory (CM, CCR7+CD45RA−), effector memory (EM, CCR7−CD45RA−), and terminally differentiated effector memory (TEMRA, CCR7− CD45RA+) T cells.17 (link) Staining was performed using the following antibodies as previously described:18 (link) CD45-V500, CD3-PerCP-Cy5.5, CD8-APCH7, CCR7-FITC, CD45RAPE (all BD Biosciences, San Jose, CA, USA) and CD4-eFluor450 (eBioscience, San Diego, CA, USA) (Online Supplementary Figure S1). Immunophenotyping was performed using a BD FACSCanto II (BD Bioscience, San Jose, CA, USA).
6
Immunophenotyping of Peripheral Blood Cells
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Samples were prepared for cytometric analysis from freshly collected PB. The samples were incubated with the following set of monoclonal antibodies: anti-CD3 FITC, anti-CD3 PECy5, anti-CD4 FITC, anti-CD8 FITC, anti-CD8 PE, anti-CD19 FITC, anti-CD19 PE, anti-CD25 PECy5, CD45RA PE, CD45RO PE, and anti-CD3 FITC/anti-CD16 PE/anti-CD56 PE (BD Pharmingen, USA). Erythrocytes were removed from the samples by adding a lysing solution (FACS Lysing Solution, Becton Dickinson, USA). The immunophenotype of the PB cells was determined using a FACSCalibur flow cytometer (Becton Dickinson, USA) equipped with a 488-nm argon laser. The results were analyzed with CellQuest Pro software (Becton Dickinson, USA).26 ,31
7
Multicolor Flow Cytometry of PBMCs
8
Lymphocyte Subset Analysis by Flow Cytometry
9
Comprehensive Immunophenotyping Protocol
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Chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse or anti-human fluorochrome labeled antibodies (Abs): CD4-PB, CD62L-APC, CD44-PerCP-Cy5.5, CD127-PE, FoxP3-FITC, Tim3-PE, PD1-PECy7, IFN-γ-PECy7, TNFα-PerCPCy5.5, IL-17-PerCPCy5.5, CD25APC-Cy7, CD45RA-PE, CD45RO-APC, and Abs for ELISA were procured from BD Pharmingen (San Diego, CA) or otherwise mentioned. RPMI-1640 and FBS were purchased from GIBCO (Grand Island, NY) for cell culture. For culturing of cells, tissue culture grade plastic-wares were purchased from BD Biosciences (Bedford, MA). Ab against iNOS used in Western blot was procured from (Abcam, Cambridge, United Kingdom).
10
Monitoring Donor Chimerism Post-Transplantation
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Peripheral blood samples were taken at day 7, 21, 35, and 63 days post-transplantation from the external jugular vein. Additional blood samples were taken at clinical signs of rejection. For assessment of ACI donor chimerism, combinations of conjugated mouse anti-rat RT1a-FITC (for MHC class I of donor cells, clone C3, BD Pharmingen) with CD4-PE (clone OX-35), CD8a-PE (clone OX-8), and CD45RA-PE (clone OX-33) were used. After incubation, a lysing solution was used, and then samples were fixed with a 1% paraformaldehyde (PFA) solution. Opposing control panels were tested and included isotype-matched antibodies IgG1-FITC/IgG2-PE and PBS samples. The analysis was performed on 1 × 104 cells, using FACS SCAN (BD Pharmingen) and FlowJo software (Siemionow et al. 2005b (link), 2008 (link)).
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