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Rosettesep human t cell enrichment kit

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The RosetteSep Human T Cell Enrichment Kit is a negative selection kit used to enrich T cells from human peripheral blood mononuclear cells (PBMCs). It uses a cocktail of antibodies that bind to non-T cells, allowing the T cells to be separated by a density centrifugation step.

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Lab products found in correlation

Cryostor cs10 cryopreservation medium, by STEMCELL (5 mentions) Human peripheral blood leukopak, by STEMCELL (1 mentions) Cryostor cs10 medium, by STEMCELL (1 mentions) Sepmate 50 tube, by STEMCELL (1 mentions) Lymphoprep density gradient medium, by STEMCELL (1 mentions) Cryostor cs10 cryopreservation media, by STEMCELL (1 mentions) Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions) Retronectin, by Takara Bio (1 mentions) Anti pe microbeads, by Miltenyi Biotec (1 mentions) Ficoll, by Merck Group (1 mentions) Leukopak, by STEMCELL (1 mentions) Imdm 10 ab human serum, by Thermo Fisher Scientific (1 mentions) Percoll, by GE Healthcare (1 mentions) Leukapheresis packs, by STEMCELL (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) β mercaptoethanol, by Merck Group (1 mentions)

11 protocols using rosettesep human t cell enrichment kit

1

Isolation and Cryopreservation of Primary Human T Cells

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Healthy donor buffy coats were collected by and purchased from the Stanford Blood Center under an IRB-exempt protocol. Primary human T cells were isolated using the RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) according to the manufacturer’s protocol using Lymphoprep density gradient medium and SepMate-50 tubes. Isolated T cells were cryopreserved at 2×107 T cells per vial in CryoStor CS10 cryopreservation medium (Stem Cell Technologies).
Lynn R.C., Weber E.W., Sotillo E., Gennert D., Xu P., Good Z., Anbunathan H., Lattin J., Jones R., Tieu V., Nagaraja S., Granja J., DeBourcy C., Majzner R., Satpathy A.T., Quake S.R., Monje M., Chang H, & Mackall C.L. (2019). c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature, 576(7786), 293-300.
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2

Isolation and Cryopreservation of PBMCs and CBMCs

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PBMCs and cord blood mononuclear cells (CBMCs) were isolated from peripheral blood samples using standard Ficoll-Paque separation, cryopreserved in 90% FBS plus 10% DMSO and stored in vapor-phase liquid nitrogen. T cells were enriched from cord blood using the RosetteSep human T cell enrichment kit (Stem Cell Technologies).
Thomson Z., He Z., Swanson E., Henderson K., Phalen C., Zaim S.R., Pebworth M.P., Okada L.Y., Heubeck A.T., Roll C.R., Hernandez V., Weiss M., Genge P.C., Reading J., Giles J.R., Manne S., Dougherty J., Jasen C.J., Greenplate A.R., Becker L.A., Graybuck L.T., Vasaikar S.V., Szeto G.L., Savage A.K., Speake C., Buckner J.H., Li X.J., Bumol T.F., Wherry E.J., Torgerson T.R., Vella L.A., Henrickson S.E., Skene P.J, & Gustafson C.E. (2023). Trimodal single-cell profiling reveals a novel pediatric CD8αα+ T cell subset and broad age-related molecular reprogramming across the T cell compartment. Nature Immunology, 24(11), 1947-1959.
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3

Isolation and Cryopreservation of Human T Cells

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T cells were isolated from buffy coats purchased from the Stanford Blood Center
under an Institutional Review Board exempt protocol. Negative selection using a
RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) and SepMate-50
tubes was performed to purify primary human T cells. T cells were cryopreserved
in CryoStor CS10 medium at a concentration of 1 × 107 to 2
× 107 cells/ml.
Grosskopf A.K., Labanieh L., Klysz D.D., Roth G.A., Xu P., Adebowale O., Gale E.C., Jons C.K., Klich J.H., Yan J., Maikawa C.L., Correa S., Ou B.S., d’Aquino A.I., Cochran J.R., Chaudhuri O., Mackall C.L, & Appel E.A. (2022). Delivery of CAR-T cells in a transient injectable stimulatory hydrogel niche improves treatment of solid tumors. Science Advances, 8(14), eabn8264.
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4

Isolation of Healthy Donor T Cells

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For experiments completed at Stanford, buffy coats from anonymous, consenting healthy donors were obtained from the Stanford University Blood Center under an University Institutional Review Board-exempt protocol or obtained from a human peripheral blood leukopak (STEMCELL Technologies). CD3+ cells were isolated using the RosetteSep Human T Cell Enrichment Kit, Lymphoprep density gradient medium and SepMate-50 tubes according to the manufacturer’s protocol (STEMCELL Technologies). For experiments completed at the Children’s Hospital of Philadelphia (CHOP), purified CD3+ healthy donor T cells were obtained from the University of Pennsylvania Human Immunology Core. All purified T cells were cryopreserved in CryoStor CS10 medium (STEMCELL Technologies).
Doan A.E., Mueller K.P., Chen A.Y., Rouin G.T., Chen Y., Daniel B., Lattin J., Markovska M., Mozarsky B., Arias-Umana J., Hapke R., Jung I.Y., Wang A., Xu P., Klysz D., Zuern G., Bashti M., Quinn P.J., Miao Z., Sandor K., Zhang W., Chen G.M., Ryu F., Logun M., Hall J., Tan K., Grupp S.A., McClory S.E., Lareau C.A., Fraietta J.A., Sotillo E., Satpathy A.T., Mackall C.L, & Weber E.W. (2024). FOXO1 is a master regulator of memory programming in CAR T cells. Nature, 629(8010), 211-218.
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5

Purification and Cryopreservation of Human T Cells

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Buffy coats from healthy donors were purchased from the Stanford Blood Center under an IRB-exempt-protocol. Primary human T cells were purified by negative selection using the RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) and SepMate-50 tubes. T cells were cryopreserved at 1–2×107 cells per mL in CryoStor CS10 cryopreservation media (Stem Cell Technologies) until use.
Labanieh L., Majzner R.G., Klysz D., Sotillo E., Fisher C.J., Vilches-Moure J.G., Pacheco K.Z., Malipatlolla M., Xu P., Hui J.H., Murty T., Theruvath J., Mehta N., Yamada-Hunter S.A., Weber E.W., Heitzeneder S., Parker K.R., Satpathy A.T., Chang H.Y., Lin M.Z., Cochran J.R, & Mackall C.L. (2022). Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell, 185(10), 1745-1763.e22.
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6

Isolation and Activation of Primary Human T Cells

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Buffy coats were purchased from Stanford Blood Center under an IRB-exempt protocol, and processed using Lymphoprep density gradient medium and SepMate-50 tubes following manufacturer's instructions. Primary human T cells were positively selected using the RosetteSep Human T cell Enrichment kit (STEMCELL Technologies), and cryopreserved at 1–2 × 107 cells/vial in CryoStor CS10 cryopreservation medium (STEMCELL Technologies). Cryopreserved cells were thawed and activated the same day with CD3/CD28 Dynabeads (Gibco) at a 3:1 bead:cell ratio in T-cell media (RPMI1640 supplemented with 10% FBS, 10 mmol/L HEPES, 2 mmol/L GlutaMAX, 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 IU/mL IL2). Activated T cells were retrovirally transduced with CD93 CAR or cotransduced with CD93 CAR and CD19 iCAR on days 3 and 4 on Retronectin (Takara)-coated plates, and anti-CD3/CD28 beads were removed on day 5. Media and IL2 were changed every 2 to 3 days until day 10 or 11, when T cells were used for assays.
Richards R.M., Zhao F., Freitas K.A., Parker K.R., Xu P., Fan A., Sotillo E., Daugaard M., Oo H.Z., Liu J., Hong W.J., Sorensen P.H., Chang H.Y., Satpathy A.T., Majzner R.G., Majeti R, & Mackall C.L. (2021). NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity. Blood Cancer Discovery, 2(6), 648-665.
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7

Isolation and Cryopreservation of Primary Human T Cells

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Healthy donor buffy coats were collected by and purchased from the Stanford Blood Center under an IRB-exempt protocol. Primary human T cells were isolated using the RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) according to the manufacturer’s protocol using Lymphoprep density gradient medium and SepMate-50 tubes. Isolated T cells were cryopreserved at 2×107 T cells per vial in CryoStor CS10 cryopreservation medium (Stem Cell Technologies).
Lynn R.C., Weber E.W., Sotillo E., Gennert D., Xu P., Good Z., Anbunathan H., Lattin J., Jones R., Tieu V., Nagaraja S., Granja J., DeBourcy C., Majzner R., Satpathy A.T., Quake S.R., Monje M., Chang H, & Mackall C.L. (2019). c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature, 576(7786), 293-300.
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8

Isolation and Cryopreservation of CD39-Negative T Cells

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Healthy donor buffy coats were purchased from the Stanford Blood Center under an IRB-exempt protocol. Primary human T cells were isolated using the RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) according to the manufacturer’s protocol. Isolated T cells were cryopreserved in CryoStor CS10 cryopreservation medium (Stem Cell Technologies).
CD39− T cells were purified using anti-PE MicroBeads (Miltenyi Biotec) and LD autoMACS (Miltenyi Biotec) columns according to the manufacturer’s protocol. Depletion efficiency was assessed by flow cytometry.
Klysz D.D., Fowler C., Malipatlolla M., Stuani L., Freitas K.A., Meier S., Daniel B., Sandor K., Xu P., Huang J., Labanieh L., Leruste A., Bashti M., Keerthi V., Mata-Alcazar J., Gkitsas N., Guerrero J.A., Fisher C., Patel S., Asano K., Patel S., Davis K.L., Satpathy A.T., Feldman S.A., Sotillo E, & Mackall C.L. (2023). Inosine Induces Stemness Features in CAR T cells and Enhances Potency. bioRxiv.
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9

Isolation and Differentiation of Primary Human Immune Cells

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Buffy coats from healthy donors were purchased from the Stanford Blood Center under an IRB-exempt-protocol. Leukopaks from healthy donors were purchased from StemCell Technologies. Primary human T cells were purified by negative selection using the RosetteSep Human T cell Enrichment kit (StemCell Technologies) and SepMate-50 tubes. T cells were cryopreserved at 2 × 10 7 cells per ml in CryoStor CS10 cryopreservation medium (StemCell Technologies) until use. Primary peripheral monocytes were purified through successive density gradients using Ficoll (Sigma-Aldrich) and Percoll (GE Healthcare). Monocytes were then differentiated into macrophages by 7-9 days of culture in IMDM + 10% AB human serum (Life Technologies).
Yamada-Hunter S.A., Theruvath J., McIntosh B.J., Freitas K.A., Lin F., Radosevich M.T., Leruste A., Dhingra S., Martinez-Velez N., Xu P., Huang J., Delaidelli A., Desai M.H., Good Z., Polak R., May A., Labanieh L., Bjelajac J., Murty T., Ehlinger Z., Mount C.W., Chen Y., Heitzeneder S., Marjon K.D., Banuelos A., Khan O., Wasserman S.L., Spiegel J.Y., Fernandez-Pol S., Kuo C.J., Sorensen P.H., Monje M., Majzner R.G., Weissman I.L., Sahaf B., Sotillo E., Cochran J.R, & Mackall C.L. (2024). Engineered CD47 protects T cells for enhanced antitumour immunity. Nature, 630(8016).
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10

Isolation and Culture of Primary Human T Cells

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Human T cells were isolated from Leukapheresis packs derived from healthy adult donors (New York Blood Center) using RosetteSep Human T cell Enrichment kit (Stemcell Technologies) and Ficoll gradient centrifugation. Complete culture media consisted of RPMI 1640 (Thermo) supplemented with 10 mM HEPES (Gibco), 10 mM L-glutamine (Gibco), 10% (v/v) fetal bovine serum (FBS; Gibco), 0.34% (v/v) β-mercaptoethanol (Sigma-Aldrich), and 10 mM penicillin-streptomycin (Gibco). Cells were frozen in 40% FBS + 10% DMSO in complete media and thawed, resting for 16 hours before use in experiments. Cells were kept under standard culture conditions (37° C, 5% CO2 / 95% air).
Yuan D.J., Shi L, & Kam L.C. (2021). Biphasic response of T cell activation to substrate stiffness. Biomaterials, 273, 120797.
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