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Rosettesep reagent

Manufactured by STEMCELL
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RosetteSep reagents are cell separation products designed to simplify the isolation of specific cell types from complex samples, such as blood or bone marrow, through negative selection. The reagents work by crosslinking unwanted cells to red blood cells, allowing the desired cells to be easily separated.

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Lab products found in correlation

Ficoll paque plus, by GE Healthcare (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions) Ficoll paque, by Cytiva (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions) G csf, by STEMCELL (1 mentions) H4230 medium, by STEMCELL (1 mentions) Easysep, by STEMCELL (1 mentions) Gm csf, by STEMCELL (1 mentions) Interleukin 3 (il 3), by STEMCELL (1 mentions) Human serum, by Miltenyi Biotec (1 mentions) Texmacs, by Miltenyi Biotec (1 mentions) Akt inhibitor 8, by Merck Group (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions)

7 protocols using rosettesep reagent

1

Isolation of Cord Blood Mononuclear Cells

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De-identified human UCB samples were acquired from University of Colorado’s ClinImmune Labs cord blood bank, or from the Medical College of Wisconsin’s tissue bank, or from the Obstetrics and Gynecology department at Meriter Hospital in Madison, WI. Blood samples were diluted with a 1:1 volume of leukocyte isolation buffer (PBS containing 2% bovine calf serum and 1 mM EDTA), and mononuclear cells were isolated by density gradient centrifugation using Ficoll-paque PLUS (GE Healthcare). If red blood cell contamination was apparent, the isolated cells were resuspended in leukocyte isolation buffer and the density gradient purification was repeated. For some experiments CBMCs were specifically depleted of T cells, or cells populations of interest (e.g., cord T cells or monocytes) were isolated using RosetteSep reagents from StemCell Technologies, or by magnetic sorting using reagents from Miltenyi Corp.
Hess N.J., S Bharadwaj N., Bobeck E.A., McDougal C.E., Ma S., Sauer J.D., Hudson A.W, & Gumperz J.E. (2021). iNKT cells coordinate immune pathways to enable engraftment in nonconditioned hosts. Life Science Alliance, 4(7), e202000999.
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2

CD8+ T Cell Proliferation Assay

Cited 1 time
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T cell-suppression assays were conducted as described.24 (link) Briefly, CD3+ T cells were enriched from source leukocytes by negative selection with Rosette Sep reagents (STEMCELL Technologies, Inc., Vancouver, BC, Canada). T cells were labelled with 1 µM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Grand Island, NY, USA) and cultured with CD3/CD28 beads (Invitrogen) for 3 days. Cells were collected, stained for CD8+ T cell markers, and fixed for flow cytometric analysis. Events were gated on CD8+ T cells and percentage of proliferation was determined based on CFSE dilution.
Ware M.B., Zaidi M.Y., Yang J., Turgeon M.K., Krasinskas A., Mace T.A., Keenan K., Farren M.R., Ruggieri A.N., Li Y., Zhang C., Chen Z., Young G.S., Elnaggar O., Che Z., Maithel S.K., Bekaii-Saab T., El-Rayes B, & Lesinski G.B. (2020). Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease. British Journal of Cancer, 123(9), 1377-1386.
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3

PBMC Isolation and T Cell Purification

Cited 2 times
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Peripheral blood mononuclear cells (PBMCs) were isolated from source leukocytes of healthy adult donors (American Red Cross, Columbus, OH) with Ficoll-Paque (Amersham, Uppsala, Sweden) as previously described [29 (link),21 (link)]. For negative selection of CD4+ and CD8+ T lymphocytes, Rosette Sep reagents were employed per manufacturer’s instructions (Stem Cell Technologies, Vancouver, BC).
Mace T.A., King S.A., Ameen Z., Elnaggar O., Young G., Riedl K.M., Schwartz S.J., Clinton S.K., Knobloch T.J., Weghorst C.M, & Lesinski G.B. (2014). Bioactive compounds or metabolites from black raspberries modulate T lymphocyte proliferation, myeloid cell differentiation and Jak/STAT signaling. Cancer immunology, immunotherapy : CII, 63(9), 889-900.
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4

Senescence Profiling of T-Cells Post-Transplant

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Patients enrolled had PBTL collected for mRNA analysis before transplant and 90-days post-transplant. CD3+ T-cells were isolated using RosetteSep reagents (StemCell Technologies, Vancouver BC). RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Valencia CA) and analyzed using a custom nanostring codeset [OSU_Senescence] which includes detectors for cellular senescence (CDKN2A/p16) and standard housekeeping genes (GUSB, HPRT1, PGK1, UBC, YWAZ). Housekeeping genes of varying overall expression levels were selected based upon their relative stability in T-cells, which was confirmed in our analyses.
Rosko A.E., Huang Y., Benson D.M., Efebera Y.A., Hofmeister C., Jaglowski S., Devine S., Bhatt G., Wildes T.M., Dyko A., Jones D., Naughton M.J., Byrd J.C, & Burd C.E. (2018). Use of a Comprehensive Frailty Assessment to Predict Morbidity in Patients with Multiple Myeloma undergoing Transplant. Journal of geriatric oncology, 10(3), 479-485.
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5

CD34+ Cell Expansion with ST7

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CD34 + cells were isolated from de-identified cord blood or adult peripheral blood and cultured in semisolid media with minimal growth factor supplements (IL-3, G-CSF, Stem Cell Technologies, Vancouver B.C.), with or without ST7 or its enantiomers. Cord blood was generously provided by Dr. Ludy Dobrila from the National Cord Blood Program (Howard P. Milstein Cord Blood Center, New York Blood Center, New York, NY). Briefly, CD34+ cells were enriched by negative selection of non-CD34+ cells using specific antibodies (RosetteSep reagent, Stem Cell Technologies, Vancouver, BC, Canada), using a Ficoll-paque density gradient, and further enriched by positive selection using EasySep (Stem Cell Technologies). The enriched cells were cultured in H4230 medium (Stem Cell Technologies) containing 1% Methylcellulose in Iscove’s MDM, 30% fetal bovine serum, 2 mM L-glutamine, 1% Bovine Serum Albumin and 10–4 M β-mercaptoethanol, with GM-CSF (20 ng/ml) and IL-3 (20 ng/ml). Cells were cultured in a humidified atmosphere with 5% CO2, at 37°C and CFU-GM colonies were enumerated on day 14; treated cultures were compared to untreated controls from the same subject. Statistical tests were performed using the GraphPad Prism program; a level of 0.05 was considered significant.
Faller D.V., Castaneda S.A., Zhou D., Vedamony M., Newburger P.E., White G.L., Kosanke S., Plett P.A., Orschell C.M., Boosalis M.S, & Perrine S.P. (2016). An oral Hemokine™, α-methylhydrocinnamate, enhances myeloid and neutrophil recovery following irradiation in vivo. Blood cells, molecules & diseases, 63, 1-8.
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6

Enriched T-cell Manufacture for CAR-T Therapy

Cited 1 time
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Healthy donor-derived enriched CD4+/8+ T cells were isolated from leukocyte cones using SepMate, RosetteSep reagent (Stemcell Technologies) and Ficoll Paque density grade centrifugation. Excess cryopreserved leukapheresis from B-ALL patients on the ALLCAR19 trial (NCT02935257) were thawed, rested overnight in TexMACS (Miltenyi Biotec) supplemented with 3% human serum (Sigma Aldrich, Life Science Production) and enriched for CD4+/8+T cells using a microbead-based pan T-cell isolation kit (Miltenyi Biotec). Activation/transduction at small-scale was designed to mimic the cGMP Miltenyi CliniMACS Prodigy-based CAR-T manufacture process on ALLCAR19.20 (link) Selected T-cells were maintained in TexMACS supplemented with 3% human serum and 10 ng/mL IL7/IL15 (cTexMACS). Cells were activated with TransAct (Miltenyi Biotec) and transduced with concentrated lentivirus on retronectin-coated plates at a multiplicity of infection of 5, cells were maintained for a further 4 days for a total manufacturing time of 8 days. AKT inhibitor VIII (Merck Millipore/Ardena) was added at T-cell activation at a concentration of 1–5 µM and maintained throughout the culture period, with a media change every 48 hours.
Mehra V., Agliardi G., Dias Alves Pinto J., Shafat M.S., Garai A.C., Green L., Hotblack A., Arce Vargas F., Peggs K.S., van der Waart A.B., Dolstra H., Pule M.A, & Roddie C. (2023). AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival. Journal for Immunotherapy of Cancer, 11(9), e007002.
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7

Isolation and Culture of CLL B Cells

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The Ohio State University Institutional Review Board reviewed and approved the protocol for acquisition of CLL samples used in these laboratory studies. All patients gave written informed consent prior to any sample being collected. All patients had CLL as defined by the IWCLL 2008 criteria [27 (link)]. CLL B cells were selected using Rosette-Sep reagent (Stem Cell Technologies, Vancouver, BC, Canada) and isolated by Ficoll density gradient centrifugation (Ficoll-Paque Plus, Amershan Biosciences, Piscataway, NJ). Cells were suspended in RPMI 1640 medium (Gibco, Carlsbad, CA) supplemented with 10% heat-inactivated human serum, 2 mmol/l L-glutamine (Invitrogen, Carlsbad, CA), and 100 U/ml penicillin/100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO). Cells were incubated at 37°C in an atmosphere of 5% CO2.
Browning R.L., Mo X., Muthusamy N, & Byrd J.C. (2015). CpG oligodeoxynucleotide CpG-685 upregulates functional interleukin-21 receptor on chronic lymphocytic leukemia B cells through an NF-κB mediated pathway. Oncotarget, 6(18), 15931-15939.
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