CD4 T cells were purified using the Dynabeads FlowComp mouse CD4 kit (Life Technologies, Carlsbad, CA). Cells were then stained for CD25 expression (BD Pharmingen) and sorted by the Tufts Flow Cytometry Core. In indicated experiments, anti-human CD2 (RPA-2.10, BD Pharmingen) was used as a pseudomarker for FoxP3 expression. Isolated cells were counted and cultured with CD4+ T cells from naïve ApoE−/− mice on high-fat diet, or control C57BL/6 mice on normal chow labeled with CFSE. Cells were stimulated with 1 mg/mL anti-CD3 and anti-CD28 for 72 h and proliferation was assessed by CFSE dilution detected by flow cytometry and expressed as the Division Index for responding cells. Division Index is the average number of cell divisions that a cell in the original population has undergone and includes for cells that never divided (i.e. includes the undivided peak). Thus, it is a measure of proliferation that accurately accounts for cells that fail to divide.
Rpa 2
Manufactured by BD
The RPA-2.10 is a laboratory equipment designed for repetitive pipetting tasks. It features an automated pipetting mechanism that can precisely dispense and aspirate liquids across multiple samples. The device is capable of handling a wide range of liquid volumes, making it suitable for various applications in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads flowcomp mouse cd4 kit, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Cd16 3g8, by BD (1 mentions)
Mem 56, by Thermo Fisher Scientific (1 mentions)
Rpa t4, by BD (1 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Anti tgf β mab 1d11, by R&D Systems (1 mentions)
Il 17a tc11 18h10.1, by BioLegend (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Ulbp 1 170818, by R&D Systems (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
5 protocols using rpa 2
1
Proliferation Assay for CD4+ T Cells
2
Multicolor Flow Cytometry for Cell Subset Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleen, draining and non-draining lymph nodes were labeled directly with fluorescently conjugated antibodies. Four-color analysis was performed using a FACS Calibur (BD) with dual laser (488 and 633 nm) excitation. The analysis gate was set on the forward and side scatters to eliminate cell debris and dead cells. For cell isolation, cells were purified from pooled spleens and lymph nodes using a 70-μm cell strainer. Cells were stained using mouse monoclonal anti-hCD2 PE conjugated antibodies RPA-2.10 (BD Pharmingen), rat anti-CD4 APC conjugated antibodies RMA4–5 (BD Pharmingen), and hamster anti-TCR PerCP conjugated H57-597 (BD Pharmingen) conjugated antibodies. Single stain samples were also prepared for MoFlo calibration before cell sorting. Cells were MoFlo cell sorted as per manufacturer’s guidelines.
3
Immunological Effects of Abatacept in URD HCT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A secondary objective of this study was to assess the immunological effects of Aba. Longitudinal flow cytometry analysis included the enumeration of natural killer (NK) cells (CD3−/CD20−/CD16+/CD56hi/lo) and CD20−/CD3+/CD4+/CD8− and CD20−/CD3+/CD4−/CD8+ T cells, including naïve (CCR7+/CD45RA+), central memory (CCR7+/CD45RA−), effector memory (CCR7−/CD45RA−), and terminal effector memory subsets (CCR7−/CD45RA+). The antibody clones used for the flow cytometric analysis are as follows: CD3 (UCHT1; BD, Franklin Lakes, NJ), CD20 (H147; Invitrogen, Grand Island, NY), CD16 (3G8; BD), CD56 (B159; BD), CD8 (RPA-T8; eBioscience, San Diego, CA), CD4 (RPA-T4; BD), CD2 (RPA-2.10; BD), CD45RA (MEM-56; Invitrogen), and CCR7 (3D12; BD).
Immune studies were compared in patients <21 years treated on the separate phase 2 Aba URD HCT trial for malignant diseases (Aba2).19 (link) Patients from the Aba2 trial received majority myeloablative conditioning (MAC) and received standard 2-drug GVHD prophylaxis for a malignant disease cohort (MTX and CSA), combined with Aba. Matched (8/8) recipients were randomized between Aba and placebo and mismatched (7/8) recipients received Aba.
Immune studies were compared in patients <21 years treated on the separate phase 2 Aba URD HCT trial for malignant diseases (Aba2).19 (link) Patients from the Aba2 trial received majority myeloablative conditioning (MAC) and received standard 2-drug GVHD prophylaxis for a malignant disease cohort (MTX and CSA), combined with Aba. Matched (8/8) recipients were randomized between Aba and placebo and mismatched (7/8) recipients received Aba.
4
Detailed Immune Cell Phenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies to CD3ε (145-2C11), CD28 (37.51), CD11c (HL3), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CTLA4 (UC10-4B9), IL-4 (11B11), IL-4 (BDV4-1D11), and IFN-γ (XMG1.2), human NGFR (C40-1457), and human CD2 (RPA-2.10) were purchased from BD Biosciences. Antibodies to CD8α (53–6.7), CD16/32 (93), CD25 (PC61 and 7D4), B220 (RA3-6B2), CD45.1 (A20), CD45.2 (104), FR4 (12A5), IL-6 (MP5-20F3), and IL-17A (TC11-18H10.1) were purchased from BioLegend. Antibodies to Foxp3 (FJK-16s) and Helios (22F6) were purchased from eBioscience. Anti-TGF-β mAb (1D11) and anti–IL-2 mAb (JES6-1A12) were purchased from R&D Systems. Cyclosporin A was purchased from Sigma-Aldrich.
5
Comprehensive Immunophenotyping of Hematologic Malignancies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monoclonal antibodies (mAbs) were used to define blast populations as follows: AML: CD45 (BD, Hl30), CD34 (BD, 581/CD34), CD117 (Invitrogen,104D2), HLA-DR (BD, G46-6) and CD33 (Biolegend, WM53). T-ALL: CD45, CD5 (Biolegend, L17F12), CD7 (BD, M-T701), CD2 (BD, RPA-2.10), CD3 (BD, SK7) and CD8 (BD, RPA-T8). The following mAbs were used to define T cell subsets: CD3(BD, UCHT1), CD4 (BD, RPA-T4), CD8 (BD, RPA-T8)), CD45RO (BD, UCHL1), CD62L (BD, DREG-56), CD95 (Biolegend, DX2). RhNKG2D Fc chimera (R&D, 12990NK-050) and rhIgG1 Fc (R&D, 110-HG-100) were PE-conjugated per manufacturer instructions (Abcam) and titrated prior to use. For individual NKG2D-ligand detection the following mAbs were used: MICA (MBL, AMO1), MICB (MBL, BMO1) (both conjugated per manufacturer instructions, Abcam), ULBP-1 (170818), ULBP2/5/6 (165903), ULBP3 (166510), ULBP4 (709116) (all R&D). Cells were washed and stained in phosphate-buffered saline supplemented with 2% fetal calf serum (Gibco) at 4°C after blocking with FcR blocking reagent (Miltenyi). LIVE/DEAD® Fixable Aqua Dead Cell Stain (Invitrogen, L34966) was used for live/dead staining. When applicable, cells were fixed using Cytofix kits (BD). Flow cytometry was performed using 4-Laser M Fortessa Analyzers (BD). Flow cytometric analysis was performed using FlowJo V10 (Tree Star).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!