In vitro studies with CAR-T cells (CD19-CD28z and CD19-BBz) were performed for the assessment of efficacy and cytotoxic capacity. For that purpose, anti-CD19-expressing CAR-T cells and CD19-expressing RAJI cells were cultured for 24 h, 7 days, and 14 days (CAR-T: RAJI; 1:1, 5:1, 10:1). Anti-cancer profiling studies on days 14 and 21 were analyzed using CD3-PC7 (Beckman Coulter, 6607100), CD4-APC-A700 (Beckman Coulter, B10824), CD8-PC5.5 (Beckman Coulter, B21205), EGFR-A488 (R&D Systems, FAB10951G), CD19-ECD (Beckman Coulter, A07770), CD25-APC (Miltenyi Biotech, 130-113-284), and CD107a (LAMP-1) -PE (Miltenyi Biotech, 130-111-621) by Cytoflex Flow Cytometer analysis. In the co-culture experiments, the death of CD19+ RAJI cells after 48 h and the CD25 activation (IL2RA, IL-2 receptor alpha chain) and CD107a (marker for degranulation of lymphocytes) cytotoxic de-granulation biomarkers of CAR-T cells and control T cells in CD3+ T cells were analyzed by flow cytometry. Survival analysis of CAR-T cells was performed to control the cell viability of RAJI cells and CAR-T cells. Trypan blue (Biological Industries, #03-102-1B) was applied to identify and count surviving cells. Cell counting and viability analysis were performed with the BIO-RAD TC20 Automated Cell Counter.
Cd25 apc
Manufactured by Miltenyi Biotec
Sourced in Germany
CD25-APC is a fluorochrome-conjugated antibody that binds to the CD25 antigen, also known as the interleukin-2 receptor alpha chain. It is commonly used in flow cytometry applications for the identification and analysis of CD25-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (2 mentions)
Cyan adp, by Beckman Coulter (2 mentions)
Cd4 pe vio770, by Miltenyi Biotec (2 mentions)
Cd127 fitc, by Miltenyi Biotec (2 mentions)
Cd4 fitc, by Miltenyi Biotec (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Cd107a pe cy5, by Miltenyi Biotec (2 mentions)
Cd3 pe, by Miltenyi Biotec (2 mentions)
Cd56 fitc, by Miltenyi Biotec (2 mentions)
Macsquant flow cytometry, by Miltenyi Biotec (2 mentions)
Cd8 vioblue, by Miltenyi Biotec (2 mentions)
Cd4 viogreen, by Miltenyi Biotec (2 mentions)
Cd19 pe cy7, by Miltenyi Biotec (2 mentions)
Tc20 automated cell counter, by Bio-Rad (1 mentions)
Cd3 pc7, by Beckman Coulter (1 mentions)
Cd4 apc a700, by Beckman Coulter (1 mentions)
Egfr a488, by R&D Systems (1 mentions)
Cd8 pc5, by Beckman Coulter (1 mentions)
Trypan blue, by Sartorius (1 mentions)
Cd19 ecd, by Beckman Coulter (1 mentions)
11 protocols using cd25 apc
1
In Vitro Assessment of CAR-T Cell Efficacy
2
Immune Cell Profiles in Pregnancy and Multiple Sclerosis
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CD4+ and CD8+ T lymphocytes, Treg, NKbright and M2 monocytes were evaluated in blood specimen of HC and MS patients at different trimester of pregnancy. In addition, the same populations were analyzed in decidual tissues. The analyses were performed by a biologist blinded to the clinical data.
Non-specific sites of 3 × 106 cells were blocked with rabbit immunoglobulins G (IgG, Sigma-Aldrich), and cells were then incubated with fluorochrome-conjugated monoclonal Ab (mAb) and isotype–matched negative controls for 10 min at 4°C. The following anti-human mAbs were used: CD163 Phycoerythrin (PE) and CD14 Fluorescein isothiocyanate (FITC) for M2 monocytes, CD3 allophycocyanin (APC)-Vio770, CD16 FITC and CD56 APC for NK cells, CD3 APC-Vio770, CD4 PE-Vio770 and CD8 FITC for T cells, CD4 PE-Vio770, CD25 APC, and CD127 FITC for Treg (Miltenyi Biotec, Bergisch Gladbach, Germany). Living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light-scatter properties to exclude cell debris. Samples were analyzed using a CyAn ADP, running Summit 4.3 software (Beckman Coulter, Brea, CA, USA). The gating strategy is shown in theFigure 1 .
Non-specific sites of 3 × 106 cells were blocked with rabbit immunoglobulins G (IgG, Sigma-Aldrich), and cells were then incubated with fluorochrome-conjugated monoclonal Ab (mAb) and isotype–matched negative controls for 10 min at 4°C. The following anti-human mAbs were used: CD163 Phycoerythrin (PE) and CD14 Fluorescein isothiocyanate (FITC) for M2 monocytes, CD3 allophycocyanin (APC)-Vio770, CD16 FITC and CD56 APC for NK cells, CD3 APC-Vio770, CD4 PE-Vio770 and CD8 FITC for T cells, CD4 PE-Vio770, CD25 APC, and CD127 FITC for Treg (Miltenyi Biotec, Bergisch Gladbach, Germany). Living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light-scatter properties to exclude cell debris. Samples were analyzed using a CyAn ADP, running Summit 4.3 software (Beckman Coulter, Brea, CA, USA). The gating strategy is shown in the
3
Multicolor Flow Cytometry of PBMC
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We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
4
Flow Cytometry Analysis of Immune Cells
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Antibodies and reagents used for flow cytometry were as follows: CD11c-BV421 (585452), MHCII-fluorescein isothiocyanate (FITC) (553623), 7-aminoactinomycin D (7AAD) (51-68981E), CD69-FITC (553236), CD11c-phycoerythrin (PE) (557401), and anti-mouse CD16/CD32 (Fc block) (553142) provided by Becton Dickinson (BD); CD40-allophycocyanin (APC) (20-8050-U025), CD44-V450 (75-0441-U025), CD4-PECy7 (60-0041-U100), CD45.1-PerCPCy5 (65-0453-U500), CD3-FITC (35-0031-U500), CD8-PECy7 (60-0081), and Ghost Red 780 Viability Dye (13-0865-T100) from Tonbo Biosciences; CD4-FITC (130-109-498), CD62L-PerCP-Vio700 (130-107-072), CD25-APC (130-109-052), CD4-PerCPCy5.5 (130-109-497), and CD11c-FITC (130-102-466) provided by Miltenyi; CCR7-PE (120105) and TCRβ-APCCy7 (109220) from BioLegend; and AbC RH capture beads (A10389) and LIVE/DEAD Fixable Yellow Dead Cell Stain (L34968) from Invitrogen. In addition, cell dyes 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR) (C2927) and carboxyfluorescein diacetate succinimidyl ester (CFSE) (C34554) were obtained from Thermo Fisher Scientific, and recombinant murine chemokine MIP-3β (CCL19) (250-27B) was provided by Peprotech.
5
Quantifying Immune Cell Subsets in Spleen
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To test the cell count of CD3+CD25+ cells,CD3+CD8+ cells, CD4+CD25+ Foxp3+Tregs, CD11c+MHC-II+ DCs in the spleen, splenic single-cell suspensions were prepared with the final concentration of 1 × 107/ml before immunofluorescent staining. Fluorescent antibodies, including CD3ε-FITC, CD8a-PerCP, CD4-PE, CD25-APC, Foxp3+-FITC, CD11c-PE, MHC-II-FITC (Miltenyi Biotec, Germany), were added to the suspensions respectively. After incubation for 10mins, cells were washed in PBS, followed by centrifuging for 10 mins and fixation in 1% polyoxymethylene for more than 2 hours. Cells were analyzed using flow cytometry.
6
Regulatory T-Cell Levels in Autoimmune Diseases
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Regulatory T-cell levels were evaluated in 29 HTE, 17 HTI, 22 MS, 12 MS + HT, and 33 HC. After gentle thawing at 37°C, PBMCs were immediately added to 5-mL RPMI 1640 (Thermo Fisher Scientific), supplemented with 10% FBS (Thermo Fisher Scientific) and centrifuged to remove DMSO (Sigma-Aldrich). Samples were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and counted for flow-cytometry experiments.
For Treg evaluation, PBMCs were incubated for 5 min at 4°C with rabbit immunoglobulins G (IgG, Sigma-Aldrich) to block non-specific sites and then for 10 min at 4°C with fluorochrome-conjugated monoclonal Ab (mAb) or isotype-matched negative controls. For Treg determination the following antihuman mAbs were used: CD4 PE-Vio770, CD25 APC, and CD127 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany). Living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light-scatter properties to exclude cell debris. Samples were analyzed using a CyAn ADP, running Summit 4.3 software (Beckman Coulter, Brea, CA, USA).
For Treg evaluation, PBMCs were incubated for 5 min at 4°C with rabbit immunoglobulins G (IgG, Sigma-Aldrich) to block non-specific sites and then for 10 min at 4°C with fluorochrome-conjugated monoclonal Ab (mAb) or isotype-matched negative controls. For Treg determination the following antihuman mAbs were used: CD4 PE-Vio770, CD25 APC, and CD127 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany). Living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light-scatter properties to exclude cell debris. Samples were analyzed using a CyAn ADP, running Summit 4.3 software (Beckman Coulter, Brea, CA, USA).
7
Isolation of Human Immune Cell Subsets
8
Expansion of Regulatory T Cells
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Flow cytometry was performed using a FACSCalibur TM flow cytometer (BD, USA) equipped with CellQuest Pro Software. Isolated cells were identified based on the forward scatter and side scatter. Briefly, cells were washed and stained with CD4-FITC, CD25-APC, and CD127-PE (Miltenyi Biotech, Germany) for 30 min at 4°C. Appropriate isotype control antibodies were used for each sample to assign gates and analysis carried out, using the FlowJo software (TreeStar Inc, OR, USA).
Treg Expansion 1 × 10 5 isolated Tregs were cultured in 100 μL TexMACS TM good manufacturing practice (GMP) cell culture medium (Miltenyi Biotech, Germany) supplemented with 10% FBS (PAN-Biotech, Germany), 100 nM rapamycin (Rapamune ® , Wyeth, USA), 500 IU/mL human recombinant IL-2 and containing 50 IU/mL pen-strep (PAN-Biotech, Germany), 1 mM sodium pyruvate (Sigma-Aldrich, USA), 25 mM HEPES (Sigma-Aldrich, USA), and activated with anti-CD3/CD28-coated beads (1:4 cell: bead ratio, Treg Expansion Kit; Miltenyi Biotec) in 48 well-plate (Sigma-Aldrich, USA) for 5 days [2, 21] . Confluent wells were split into new plates, and 100 IU/mL rIL-2 was added at day 1 and replenished every second day.
Treg Expansion 1 × 10 5 isolated Tregs were cultured in 100 μL TexMACS TM good manufacturing practice (GMP) cell culture medium (Miltenyi Biotech, Germany) supplemented with 10% FBS (PAN-Biotech, Germany), 100 nM rapamycin (Rapamune ® , Wyeth, USA), 500 IU/mL human recombinant IL-2 and containing 50 IU/mL pen-strep (PAN-Biotech, Germany), 1 mM sodium pyruvate (Sigma-Aldrich, USA), 25 mM HEPES (Sigma-Aldrich, USA), and activated with anti-CD3/CD28-coated beads (1:4 cell: bead ratio, Treg Expansion Kit; Miltenyi Biotec) in 48 well-plate (Sigma-Aldrich, USA) for 5 days [2, 21] . Confluent wells were split into new plates, and 100 IU/mL rIL-2 was added at day 1 and replenished every second day.
9
Dornase alfa and MgSO4 Effects on PBMC
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Three healthy adult blood was obtained from Acibadem Labcell Cellular Therapy Laboratory.
Following the isolation of peripheral mononuclear cells (PBMC) by overlaying blood on Ficollpaque plus (GE Healthcare), the serially diluted (300 U/mL, 100 U/mL, 30 U/mL, 10 U/mL, 3 U/mL) Dornase alfa (Pulmozyme®, Roche, USA) and 420 g/mL MgSO 4 (Magnesium Sulphate 15% Onfarma, TR) were incubated with the PBMCs for 48hr in T cell medium (6% Human AB Serum and 1% Pen/Strep, Texmacs medium). Immune cell subtypes and their activation levels were determined by Miltenyi MACSQuant flow cytometry analysis using CD3-PE, CD19-PE.cy7, CD56-FITC, CD4-Viogreen, CD8-Vioblue, CD25-APC and CD107a-PE.cy5.5 (Miltenyi).
Following the isolation of peripheral mononuclear cells (PBMC) by overlaying blood on Ficollpaque plus (GE Healthcare), the serially diluted (300 U/mL, 100 U/mL, 30 U/mL, 10 U/mL, 3 U/mL) Dornase alfa (Pulmozyme®, Roche, USA) and 420 g/mL MgSO 4 (Magnesium Sulphate 15% Onfarma, TR) were incubated with the PBMCs for 48hr in T cell medium (6% Human AB Serum and 1% Pen/Strep, Texmacs medium). Immune cell subtypes and their activation levels were determined by Miltenyi MACSQuant flow cytometry analysis using CD3-PE, CD19-PE.cy7, CD56-FITC, CD4-Viogreen, CD8-Vioblue, CD25-APC and CD107a-PE.cy5.5 (Miltenyi).
10
Comprehensive Immune Cell Profiling by Flow Cytometry
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MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for flow cytometry analysis. To measure cell surface markers, Tregs or PBMCs were stained with CD4-PerCp, CD25-PE, CD127-Vio770, CD152-APC, CD39-FITC and CD223-APC antibodies (all from Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's protocol. To measure FoxP3 and Helios protein content, PBMCs or eTregs were treated with CD4-FITC, CD25-APC (all from Miltenyi Biotec, Bergisch Gladbach, Germany), FoxP3-PE or Helios-PE antibodies (BD Pharmingen, San Diego, CA, USA) followed by flow cytometry analysis according to the manufacturer's protocol. Mean fluorescence intensity (MFI) was used to determine intracellular protein expression of FoxP3 and Helios. To measure cell proliferation, isolated CD4 + cells were labeled with the vital dye CFSE (5-(and 6-)carboxyfluorescein diacetate succinimidyl ester, eBioscience Inc., San Diego, CA, USA) and analyzed by flow cytometry every 24 h during expansion. The proportion of proliferated cells was determined as the proportion of cells with reduced CFSE signal. Alternatively, a CD4 + CD25 + regulatory T cell isolation kit, human (Miltenyi Biotec), was used to obtain pTregs as a comparison group for functional tests (FOXP3 promoter methylation, gene expression and in vitro immunosuppression assays).
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