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Cd45ra apc clone hi100

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CD45RA APC (clone HI100) is a fluorochrome-conjugated monoclonal antibody that binds to the CD45RA antigen on human cells. CD45RA is a high molecular weight isoform of the CD45 protein tyrosine phosphatase, which is expressed on the surface of most leukocytes and is involved in the regulation of T and B cell antigen receptor signaling.

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Lab products found in correlation

Countbright absolute counting beads, by Thermo Fisher Scientific (3 mentions) Cd38 apc cy7 clone hit2, by BioLegend (3 mentions) Cd197 ccr7 clone g043h7 pe, by BioLegend (2 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions) Cd90 pe cy7 clone 5e10, by BioLegend (2 mentions) Cd90 pe cy7, by BioLegend (1 mentions)

Close spelling variants of this product

CD45RA (clone HI100; CD45RA (HI100, CD45RA-APC

5 protocols using cd45ra apc clone hi100

1

T Cell Subset Identification by Flow Cytometry

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Extracellular staining and acquisition were previously described.17 (link) The following antibodies were used: CD197/CCR7 (clone G043H7) PE (353203, BioLegend, San Diego, CA, USA), CD45RA (clone HI100) APC (304111, BioLegend, San Diego, CA, USA), and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (L34966, Invitrogen, Carlsbad, CA, USA). The following gating strategy was used to assess different T cell subsets: Naïve Phenotype (CCR7+CD45RA+), Central Memory /TCM (CCR7+CD45RA−), Effector Memory Cells re-expressing CD45RA/TEMRA (CCR7−CD45RA+) and Effector Memory/TEM (CCR7−CD45RA−).
Cox M.J., Roman C.M., Tapper E.E., Siegler E.L., Chappell D., Durrant C., Ahmed O., Sinha S., Mwangi R., Scott N.S., Hefazi M., Schick K.J., Horvei P., Ruff M.W., Can I., Adada M., Bezerra E., Fonkoua L.A., Parikh S.A., Kay N.E., Sakemura R, & Kenderian S.S. (2022). GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity. Leukemia, 36(6), 1635-1645.
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2

T Cell Subset Identification by Flow Cytometry

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Extracellular staining and acquisition were previously described.17 (link) The following antibodies were used: CD197/CCR7 (clone G043H7) PE (353203, BioLegend, San Diego, CA, USA), CD45RA (clone HI100) APC (304111, BioLegend, San Diego, CA, USA), and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (L34966, Invitrogen, Carlsbad, CA, USA). The following gating strategy was used to assess different T cell subsets: Naïve Phenotype (CCR7+CD45RA+), Central Memory /TCM (CCR7+CD45RA−), Effector Memory Cells re-expressing CD45RA/TEMRA (CCR7−CD45RA+) and Effector Memory/TEM (CCR7−CD45RA−).
Cox M.J., Roman C.M., Tapper E.E., Siegler E.L., Chappell D., Durrant C., Ahmed O., Sinha S., Mwangi R., Scott N.S., Hefazi M., Schick K.J., Horvei P., Ruff M.W., Can I., Adada M., Bezerra E., Fonkoua L.A., Parikh S.A., Kay N.E., Sakemura R, & Kenderian S.S. (2022). GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity. Leukemia, 36(6), 1635-1645.
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3

Editing Hematopoietic Stem Cells

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At day 0, CD34+ HSPCs were thawed and stained with the following antibody panel: CD34 BV 421, CD38 APC-Cy7 (clone HIT2, BioLegend), CD90 PE-Cy7 (clone 5E10, BioLegend), and CD45RA APC (clone HI100, BioLegend). The cells were then FACS sorted into different subsets including CD38+ cells, HSCs and multipotent progenitors (MPPs). Unsorted cells were used as an additional cell population. After 2 days, both sorted and unsorted cells were electroporated and transduced with AAV6_PGK-GFP. At days 5 and 12 post editing, the cells were stained with the above antibody panels to determine the percentage of HSCs and MPPs as well as evaluate GFP expression by flow cytometry. Cell yields were calculated by adding 10 μL of Count Bright Absolute Counting Beads (ThermoFisher Scientific), following the manufacturer’s protocol.
Rai R., Naseem A., Vetharoy W., Steinberg Z., Thrasher A.J., Santilli G, & Cavazza A. (2023). An improved medium formulation for efficient ex vivo gene editing, expansion and engraftment of hematopoietic stem and progenitor cells. Molecular Therapy. Methods & Clinical Development, 29, 58-69.
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4

Purification and Genetic Modification of HSPCs

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At day 0, CD34+ HSPCs were thawed and stained with the following antibody panel: CD34 BV 421, CD38 APC-Cy7 (clone HIT2, BioLegend), CD90 PE-Cy7 (clone 5E10, BioLegend) and CD45RA APC (clone HI100, BioLegend). The cells were then FACS-sorted into different subsets including CD38+ cells, haematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). Unsorted cells were taken as an experimental control. After 2 days, both sorted and unsorted cells were electroporated and transduced with AAV6_PGK-GFP. At days 5  and 12  post editing, the cells were stained with the above antibody panels to determine the percentage of HSCs and MPPs as well as evaluate GFP expression by FACS. Cell yields were calculated by adding 10 microliters of Count Bright Absolute Counting Beads (ThermoFisher Scientific), following the manufacturer’s protocol.
Rai R., Romito M., Rivers E., Turchiano G., Blattner G., Vetharoy W., Ladon D., Andrieux G., Zhang F., Zinicola M., Leon-Rico D., Santilli G., Thrasher A.J, & Cavazza A. (2020). Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome. Nature Communications, 11, 4034.
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5

Tracking Edited Hematopoietic Stem Cells

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At day 0, CD34 + HSPCs were thawed and stained with the following antibody panel: CD34 BV 421, CD38 APC-Cy7 (clone HIT2, BioLegend), CD90 PE-Cy7 (clone 5E10, BioLegend) and CD45RA APC (clone HI100, BioLegend). The cells were then FACS-sorted into different subsets including CD38+ cells, haematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). Unsorted cells were taken as an experimental control. After 2 days, both sorted and unsorted cells were electroporated and transduced with AAV6_PGK-GFP. At days 5 and 12 post editing, the cells were stained with the above antibody panels to determine the percentage of HSCs and MPPs as well as evaluate GFP expression by FACS. Cell yields were calculated by adding 10 microliters of Count Bright Absolute Counting Beads (ThermoFisher Scientific), following the manufacturer's protocol.
Rai R., Vetharoy W., Naseem A., Steinberg Z., Thrasher A.J., Santilli G., & Cavazza A. (2022). Optimized cell culture conditions promote ex-vivo manipulation and expansion of primitive hematopoietic stem cells for therapeutic gene editing.
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