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Anti ccr7 fitc clone 150503

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Anti-CCR7–FITC (clone 150503) is a laboratory reagent used for the detection and analysis of the CCR7 protein, which is expressed on the surface of certain cell types. This fluorescently labeled antibody can be used in flow cytometry applications to identify and quantify CCR7-positive cells.

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Lab products found in correlation

Lsrfortessa, by BD (3 mentions) Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions) Live dead fixable violet, by Thermo Fisher Scientific (2 mentions) Anti cd3 bv605 clone okt3, by BioLegend (1 mentions) Lsrfortessa system, by BD (1 mentions) Anti cd3 cd28 beads, by Miltenyi Biotec (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Facsaria 2, by BD (1 mentions) Interleukin 2 (il 2), by STEMCELL (1 mentions) Anti cd19 pb clone hib19, by BioLegend (1 mentions)

Close spelling variants of this product

CCR7 (clone 150503), Anti-CCR7-FITC CCR7-FITC Anti-CCR7

5 protocols using anti ccr7 fitc clone 150503

1

Phenotypic Characterization of Activated CAR T Cells

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Activated CAR T cells were expanded for 9 to 11 days and subsequently stained with a panel of monoclonal antibodies to assess differentiation. The following pre-titrated antibodies were used: anti-CCR7–FITC (clone 150,503; BD PharMingen); anti-CD45RO-PE (clone UCHL1), anti-CD8-H7APC (clone SK1; BD Biosciences); anti-CD4-BV605 (clone OKT4). 1 × 106 cells were immunostained as follows: cells were washed with PBS and stained for viability using LIVE/DEAD Fixable aqua (Molecular Probes) for 15 min, washed once, and resuspended in fluorescence-activated cell sorting (FACS) buffer consisting of PBS, 1% BSA, and 5 mM EDTA. Cells were then incubated with the above indicated antibodies for 30 min at room temperature. Samples were then washed three times with FACS buffer and fixed in 1% paraformaldehyde. Positively stained cells were differentiated from background using fluorescence-minus-one controls. Flow cytometry was performed on BD LSR Fortessa. Analysis was performed using FlowJo software (Tree Star version 10.1).
Durgin J.S., Thokala R., Johnson L., Song E., Leferovich J., Bhoj V., Ghassemi S., Milone M., Binder Z., O'Rourke D.M, & O'Connor R.S. (2021). Enhancing CAR T function with the engineered secretion of C. perfringens neuraminidase. Molecular Therapy, 30(3), 1201-1214.
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2

Assessing T Cell Differentiation Status

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Activated T cells were expanded for 9–11 days and subsequently stained with a panel of monoclonal antibodies to assess differentiation status. The following pre-titrated antibodies were used: anti-CCR7–FITC (clone 150503; BD PharMingen); anti-CD45RO-PE (clone UCHL1), anti-CD8-H7APC (clone SK1; BD Biosciences); anti-CD4-BV605 (clone OKT4). 1 × 106 cells were immunostained as follows: cells were washed with phosphate-buffered saline (PBS) and stained for viability using LIVE/DEAD Fixable aqua (Molecular Probes) for 15 min, washed once, and resuspended in fluorescence activated cell sorting (FACS) buffer consisting of PBS, 1% BSA, and 5 mM EDTA. Cells were then incubated with the above indicated antibodies for 30 min at room temperature (RT). Samples were then washed three times with FACS buffer and fixed in 1% paraformaldehyde. Positively stained cells were differentiated from background using fluorescence-minus-one (FMO) controls. Flow cytometry was performed on BD LSR Fortessa. Analysis was performed using Flowjo software (Tree Star version 10.1).
Ghassemi S., Martinez-Becerra F.J., Master A.M., Richman S.A., Heo D., Leferovich J., Tu Y., García-Cañaveras J.C., Ayari A., Lu Y., Wang A., Rabinowitz J.D., Milone M.C., June C.H, & O’Connor R.S. (2020). Enhancing Chimeric Antigen Receptor T Cell Anti-tumor Function through Advanced Media Design. Molecular Therapy. Methods & Clinical Development, 18, 595-606.
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3

Comprehensive T-cell Phenotyping Protocol

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T-cell differentiation was assessed using the following antibodies: anti-CCR7–FITC clone 150503 (BD Pharmingen); anti-CD45RO–PE clone UCHL1 and anti-CD8–H7APC clone SK1 (BD Biosciences); and anti-CD4–BV510 clone OKT4, anti-CD3–BV605 clone OKT3, anti-CD14–Pacific Blue clone HCD14 and anti-CD19–Pacific Blue clone H1B19 (BioLegend). The anti-CAR19 idiotype for surface expression of CAR19 was provided by Novartis. Cells were washed with PBS, incubated with LIVE/DEAD fixable violet (Molecular Probes) for 15 min and resuspended in fluorescence activated cell sorting buffer consisting of PBS, 1% BSA and 5 mM EDTA. The cells were then incubated with antibodies for 1 h at 4 °C. Positively stained cells were differentiated from the background using fluorescence-minus-one controls. A representative gating strategy to identify T-cell subsets is shown in Supplementary Fig. 5. Flow cytometry was performed on a BD LSR Fortessa system. Analysis was performed using the FlowJo software (Tree Star Inc. version 10.1).
Ghassemi S., Durgin J.S., Nunez-Cruz S., Patel J., Leferovich J., Pinzone M., Shen F., Cummins K.D., Plesa G., Cantu V.A., Reddy S., Bushman F.D., Gill S.I., O’Doherty U., O’Connor R.S, & Milone M.C. (2022). Rapid manufacturing of non-activated potent CAR T cells. Nature Biomedical Engineering, 6(2), 118-128.
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4

T cell Subset Isolation and Stimulation

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PBMC or sorted T cell populations were stimulated with anti-CD3/CD28
beads (Miltenyi Biotec) in the presence of 20 U/ml IL-2 (Stemcell) for 72
hours in supplemented culture media (RPMI 1640 (Gibco) supplemented with
L-glutamine, 10% FCS and Penicillin/Streptomycin). Sorting was
performed on a FACS Aria II (BD Biosciences) after staining for TN
(CCR7+CD45RA+CD27+), TCM
(CD27+CD45RA−CCR7+), TEM
(CD27−CCR7−CD45RA−), TEMRA
(CD27−CCR7−CD45RA+) and PD-1+ populations
using anti-CD27-BV785 (clone O323), anti-CD45RA-BV605 (clone HI100),
anti-PD-1-BV421 (clone E12.2.H7), anti-CD8 APC-Fire (clone RPA-T8)
(Biolegend), anti-CCR7-FITC (clone 150503) (BD Biosciences), and after
staining with life/dead reagent Ghost Violet (Tonbo).
Bengsch B., Ohtani T., Khan O., Setty M., Manne S., O’Brien S., Gherardini P.F., Herati R.S., Huang A.C., Chang K.M., Newell E.W., Bovenschen N., Pe’er D., Albelda S.M, & Wherry E.J. (2018). Epigenomic-guided mass cytometry profiling reveals disease-specific features of exhausted CD8 T cells. Immunity, 48(5), 1029-1045.e5.
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5

Immunophenotyping of T-cell Differentiation

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At harvest or upon thawing, 1 × 106 cells were stained for cell surface markers to analyze T-cell differentiation status. The following pretitrated antibodies were used: anti-CCR7–FITC (clone 150503; BD Pharmingen); anti-CD45RO–PE (clone UCHL1), anti-CD8–H7APC (clone SK1; BD Biosciences); anti-CD95–PerCP-Cy5.5 (Clone DX2), anti-CD4–BV510 (clone OKT4), anti-CD3–BV605 (clone OKT3), anti-CD14–Pacific Blue (PB; clone HCD14), anti-CD19–PB (clone HIB19; BioLegend); anti-CD27–PE-Cy7 (clone 1A4CD27; Beckman Coulter); and carboxyfluorescein diacetate succinimidyl ester (CFSE) and ViViD (Invitrogen). The anti-CAR19 idiotype for surface expression of CAR19 was provided by Novartis (Basel, Switzerland).
Cells were washed with PBS and stained for viability using LIVE/DEAD Fixable Violet (Molecular Probes) for 15 minutes, washed once, and resuspended in fluorescence-activated cell sorting (FACS) buffer consisting of PBS, 1% BSA, and 5 mmol/L ethylenediaminetetraacetic acid (EDTA). Cells were then incubated with the above indicated antibodies for 1 hour at 4°C. Sample were then washed 3 times with FACS buffer and fixed in 1% paraformaldehyde. Positively stained cells were differentiated from background using fluorescence-minus-one controls. Flow cytometery was performed on BD LSR Fortessa. Analysis was performed using Flowjo software (Tree Star Inc. version 10.1).
Ghassemi S., Nunez-Cruz S., O’Connor R.S., Fraietta J.A., Patel P.R., Scholler J., Barrett D.M., Lundh S.M., Davis M.M., Bedoya F., Zhang C., Leferovich J., Lacey S.F., Levine B.L., Grupp S.A., June C.H., Melenhorst J.J, & Milone M.C. (2018). Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells. Cancer immunology research, 6(9), 1100-1109.
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