Affinity-purified F(ab′)2 fragments of polyclonal goat anti-mouse IgM (anti-Ig) and polyclonal goat anti-rabbit Ab-APC were obtained from Jackson ImmunoResearch Laboratories. Anti-mouse mAbs against CD19-APC, B220-PE, B220-FITC, CD5-PE-Cy7, CD43-PE, PD-L2-PE, and PD-L2-APC, CD86-PE, CD44-PE, CD9-PE, CD80-PE, Thy-1-PE, CD45.1-FITC, phospho-p38, and phospho-ERK-APC were obtained from BD pharmingen. Polyclonal anti-pERK, anti-NFATc1, anti-RasGRP3, and anti-phospho-JNK antibodies were obtained from Cell Signaling Technology. Anti-RasGRP1 antibody was obtained from Santa Cruz Biotechnology. Phorbol ester myristate (PMA), RIPA buffer, phenylmethylsulfonyl fluoride (PMSF), and protein inhibitor cocktails were obtained from Sigma Aldrich.
Cd5 pe cy7
Manufactured by BD
The CD5-PE-Cy7 is a fluorescently-labeled monoclonal antibody used for flow cytometry applications. It binds to the CD5 antigen, which is expressed on a subset of T cells and B cells. The PE-Cy7 fluorophore attached to the antibody allows for detection and quantification of CD5-positive cells in a sample.
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Lab products found in correlation
Cd19 pe cy5, by BD (2 mentions)
Anti ki67 fitc, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Protein inhibitor cocktail, by Merck Group (1 mentions)
Pd l2 pe, by BD (1 mentions)
Anti phospho jnk antibody, by Cell Signaling Technology (1 mentions)
Cd86 pe, by BD (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions)
B220 fitc, by BD (1 mentions)
Cd45.1 fitc, by BD (1 mentions)
Cd9 pe, by BD (1 mentions)
Phospho p38, by BD (1 mentions)
Cd43 pe, by BD (1 mentions)
Affinity purified f ab 2 fragments of polyclonal goat anti mouse igm anti ig, by Jackson ImmunoResearch (1 mentions)
Cd80 pe, by BD (1 mentions)
Cd44 pe, by BD (1 mentions)
Cd27 apc, by BD (1 mentions)
Cd45ra fitc, by BD (1 mentions)
Ccr7 pe cf594, by BD (1 mentions)
4 protocols using cd5 pe cy7
1
Comprehensive Antibody Characterization for Cell Analysis
2
Isolation and Analysis of CLL Cell Subsets
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Isolation of cell fractions on the basis of expression of CXCR4 and CD5 was carried out as previously described.16 (link) Flow cytometry reagents were purchased from BD Bioscience, San Jose, CA, USA. Briefly, cells were incubated with murine anti-human mAbs: CD5-FITC (cat# 555352), CD19-APC (cat# 555415) and CXCR4-PE (cat #555974). After gating on CD19+CD5+ events, cells were sorted with a BD FACSAria on the basis of intensity of CXCR4 and CD5 expression. Isolated cell pellets were stored at −80°C until analysis.
Ki67 expression was assessed in CLL cells stained with CD19-PE-Cy5 (cat# 555414) and CD5-PE-Cy7 (cat# 348790).18 (link) After surface staining, cells were washed and suspended in 4% paraformaldehyde (on ice, 1h), washed, resuspended in 70% ethanol (at –20°C, 2h), washed, incubated with anti-Ki67-FITC (cat# 556026, BD Biosciences) for 20min at room temperature, washed, and analyzed on a BD FACSCanto II flow cytometer. Data were analyzed using FlowJo software (Tree Star Inc.).
Ki67 expression was assessed in CLL cells stained with CD19-PE-Cy5 (cat# 555414) and CD5-PE-Cy7 (cat# 348790).18 (link) After surface staining, cells were washed and suspended in 4% paraformaldehyde (on ice, 1h), washed, resuspended in 70% ethanol (at –20°C, 2h), washed, incubated with anti-Ki67-FITC (cat# 556026, BD Biosciences) for 20min at room temperature, washed, and analyzed on a BD FACSCanto II flow cytometer. Data were analyzed using FlowJo software (Tree Star Inc.).
3
Isolation and Analysis of CLL Cell Subsets
4
Comprehensive T Cell Subset Analysis
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For T cell subset analyses, 100 μL of whole blood was mixed with an optimal dilution of the following monoclonal antibodies: CD3-APC-CY7, CD4-PE, CD8-PerCP-Cy5.5, CD45RA-FITC, CCR7-PE-CF594, CD5-PE-CY7, CD27-APC, CD127-V450, CD25-BV510 (BD Bioscience). After adding all antibodies, the mixture was gently vortexed and incubated for 15 min in the dark at room temperature. Subsequently, 2 mL of 1× FACS lysing solution (BD Bioscience) was added and incubated for 10 min in the dark at room temperature. Finally, cells were washed 3 times with 200 μL washing buffer. Data was collected by a 9-colour Aria I flow cytometer (BD Bioscience) and analyzed with FACSDiva software (version 6.1.3, BD Bioscience). The following T cell subsets were determined (Mahnke et al., 2012; Schatorje et al., 2012)
Tem), and CD8 + transitional memory T cells (CD3 + CD8 + CD4 -CD45RA -CCR7 -CD27 + , CD8 + Ttm). The full-minus-one (FMO) principle was adopted for gating the T cell subsets. Absolute counts of each subgroup were calculated by multiplying the total lymphocyte counts with the percentages of each subset that were determined by flow cytometry.
Tem), and CD8 + transitional memory T cells (CD3 + CD8 + CD4 -CD45RA -CCR7 -CD27 + , CD8 + Ttm). The full-minus-one (FMO) principle was adopted for gating the T cell subsets. Absolute counts of each subgroup were calculated by multiplying the total lymphocyte counts with the percentages of each subset that were determined by flow cytometry.
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