Easysep human na ve cd8 t cell isolation kit

Manufactured by STEMCELL

The EasySep™ Human Naïve CD8+ T Cell Isolation Kit is a cell separation product designed to isolate naïve CD8+ T cells from human peripheral blood mononuclear cells (PBMCs). The kit utilizes a straightforward, column-free magnetic separation technology to enrich the target cell population.

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Lab products found in correlation

Gentamycin, by MP Biomedicals (1 mentions) Pen strep, by Thermo Fisher Scientific (1 mentions) Easysep human t cell isolation kit, by STEMCELL (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Recombinant human il 2, by Thermo Fisher Scientific (1 mentions) Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Human ab serum, by Gemini Bio (1 mentions) Cellgro dc medium, by CellGenix (1 mentions) Transwell system, by Corning (1 mentions) Immunocult human cd3 cd28 t cell activator, by STEMCELL (1 mentions) Mr frostytm, by Thermo Fisher Scientific (1 mentions) Facsaria, by BD (1 mentions)

4 protocols using easysep human na ve cd8 t cell isolation kit

Primary T and NK Cell Isolation and Activation

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For T cell preparations, buffy coats were obtained from the New York Blood Center or, alternatively, Leukocyte Reduction System cones (STEMCELL Technologies, 200–0093). T cells were isolated by EasySep^™ Human T Cell Isolation Kits (STEMCELL Technologies, 17951). Naïve T cells were isolated by EasySep^™ Human Naïve Pan T Cell Isolation Kits (STEMCELL Technologies, 17961). Naïve CD8⁺ T cells were isolated by EasySep^™ Human Naïve CD8⁺ T Cell Isolation Kit (STEMCELL Technologies, 19258). All isolated T cells were activated by CD3/CD28 Dynabeads (Thermo Fisher, 11132D) at a 1:1 ratio for 2 days. For NK cell preparations, cord blood mononuclear cells were ordered from STEMCELL Technologies (STEMCELL Technologies, 70007.1 or 70007.2). NK cells were isolated by EasySep^™ Human NK Cell Isolation Kits (STEMCELL Technologies, 17955) and activated by 100 Gy irradiated K562 Clone 9 feeder cells. Feeder cells were mixed with NK cells at a 2:1 tumor:NK cell ratio. Primary cells were cultured in RPMI 1640 (Gibco, 11875085) with 1% v/v Penstrep (Gibco, 15070–063), 2.5% v/v HEPES (Gibco, 15630–080), 0.02% v/v Gentamycin (MP Biomedicals, 1676245) and 10% v/v heat inactivated human serum AB (GeminiBio, 100–512). Recombinant human IL-2 (PeproTech, 200–02) was added to T cells and NK cells cultures at 50 IU mL⁻¹ or 200 IU mL⁻¹, respectively.

Yi F., Cohen T., Zimmerman N., Dündar F., Zumbo P., Eltilib R., Brophy E.J., Arkin H., Feucht J., Gormally M.V., Hackett C.S., Kropp K.N., Etxeberria I., Chandran S.S., Park J.H., Hsu K.C., Sadelain M., Betel D, & Klebanoff C.A. (2024). CAR-engineered lymphocyte persistence is governed by a FAS ligand/FAS auto-regulatory circuit. bioRxiv.

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Priming Naive CD8+ T Cells with Melan-A Antigen

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The priming assay is based on a previously described protocol^{68 (link)}. Briefly, DCs were generated from plastic-adherent human monocytes isolated from PBMCs of HLA-A2 + healthy donors. After 72 h of culture in GM-CSF/IL4-containing CellGro DC medium (CellGenix, cat. no. 20801-0500) with 1% human serum (HS), DCs were matured in medium containing 10 ng/mL IL4, 800 IU/mL GM-CSF, 10 ng/mL LPS, and 100 IU/mL IFNγ. Mature DCs were cultured in the presence or absence of 2.5 µg/mL Melan-A antigen peptide (ELAGIGILTV, JPT, cat. no. SP-MHCI-0006) for 16 h. The cells were then irradiated (30 Gy) and washed. Next, autologous naive CD8+ T cells were isolated from PBMCs with the EasySEP Human Naïve CD8+ T Cell Isolation Kit (Stem cell Technologies, cat. no. 19258) and co-incubated with mature DCs at a 4:1 ratio in CellGro DC medium with 5% HS and 30 ng/mL IL-21. Test antibodies at 10 µg/mL were added on day 0 and day 3 of co-culture. Fresh medium containing 5 ng/mL IL-7 and 5 ng/mL IL-15 was added on days 3 and 5. On day 10 of co-culture, T cells were harvested, counted, and stained with CD8, CD45RA, and CCR7 antibodies and ELAGIGILTV Dextramer (Immudex, cat. no. WB2162-APC) and analyzed by FACS.

Geuijen C., Tacken P., Wang L.C., Klooster R., van Loo P.F., Zhou J., Mondal A., Liu Y.B., Kramer A., Condamine T., Volgina A., Hendriks L.J., van der Maaden H., Rovers E., Engels S., Fransen F., den Blanken-Smit R., Zondag-van der Zande V., Basmeleh A., Bartelink W., Kulkarni A., Marissen W., Huang C.Y., Hall L., Harvey S., Kim S., Martinez M., O’Brien S., Moon E., Albelda S., Kanellopoulou C., Stewart S., Nastri H., Bakker A.B., Scherle P., Logtenberg T., Hollis G., de Kruif J., Huber R., Mayes P.A, & Throsby M. (2021). A human CD137×PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade. Nature Communications, 12, 4445.

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Naïve CD8+ T-cell Recruitment Assay

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Human naïve CD8+ T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs), using EasySep™ Human Naïve CD8+ T Cell Isolation Kit (STEMCELL Technologies). Purified naïve CD8+ T cells were stimulated with ImmunoCult™ Human CD3/CD28 T Cell Activator (STEMCELL Technologies) for 72 h. CD8+ T‐cell recruitment assay was performed using Transwell system (Costar). Briefly, CD8+ T cells (2 × 10⁵/per well) activated as described above were added into the upper chamber, while culture supernatants from BC cells were added into the lower chamber. Twenty‐four hours later, cells that migrated through the micropores (5‐μm pore size) of the upper chambers into the lower chambers were collected and enumerated using a flow cytometer (BD Biosciences). All recruitment assays were performed with only one volunteer product at a time.

Song S., Zhang D., Chen J., Qi L., Zhang M., Yang X., Ye T., Ye Q, & Lin J. (2023). CHMP4A stimulates CD8+ T‐lymphocyte infiltration and inhibits breast tumor growth via the LSD1/IFNβ axis. Cancer Science, 114(8), 3162-3175.

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Isolation and Characterization of PBMCs

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All experiments were performed using peripheral blood mononuclear cells (PBMCs) isolated from buffy coats of healthy donors (Sanquin Blood Supply Foundation). For antigen-specific experiments, HLA-A2 and/or B7-positive donors were used who lack the expression of the MiHA of interest. PBMCs were isolated via Ficoll-paque^TM PLUS gradient (cat#17-1440-03, Sigma-Aldrich), followed by selection of CD14⁺ and/or CD8⁺ cells by magnetic bead isolation, on fresh and cryopreserved material respectively (cat#130-050-201 and cat#130-045-201, Miltenyi Biotec). Cryopreserved material was frozen using a Mr. Frosty^TM (cat#5100-0001, ThermoFisher) in self made serum-supplemented medium with 10% DMSO. For polyclonal T cell stimulation or allogeneic mixed lymphocyte reactions (allo-MLR), further purification of CD8⁺ naive T (T_N) cells was performed via FACS-sorting of CD3⁺CD8⁺CCR7⁺CD45RO⁻ cells using the FACS Aria (BD Bioscience). The EasySep™ Human Naïve CD8⁺ T Cell Isolation Kit (cat#19258, Stemcell Technologies) was used to isolate CD8⁺ T_N cells from fresh or cryopreserved material for T cell priming experiments. Cell numbers were based on trypan blue cell counting, and all isolations resulted in ≥90% purity.

Mousset C.M., Hobo W., Ji Y., Fredrix H., De Giorgi V., Allison R.D., Kester M.G., Falkenburg J.H., Schaap N.P., Jansen J.H., Gattinoni L., Dolstra H, & van der Waart A.B. (2018). Ex vivo AKT-inhibition facilitates generation of polyfunctional stem cell memory-like CD8+ T cells for adoptive immunotherapy. Oncoimmunology, 7(10), e1488565.

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