Easysep mouse cd90.2 positive selection kit 2

Manufactured by STEMCELL

Sourced in United States

The EasySep Mouse CD90.2 Positive Selection Kit II is a laboratory tool designed for the isolation of CD90.2-positive cells from mouse cell suspensions. The kit utilizes magnetic particles and an easy-to-use positive selection protocol to enrich for the target cell population.

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Lab products found in correlation

Mouse ifn gamma quantikine elisa kit, by R&D Systems (1 mentions) Facsaria 2, by BD (1 mentions) Easysep mouse t cell isolation kit, by STEMCELL (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Anti ifnar1 antibody, by BioXCell (1 mentions) Easysep human na ve cd4 t cell isolation kit, by STEMCELL (1 mentions) Easysep mouse na ve cd4 t cell isolation kit, by STEMCELL (1 mentions) Histopaque, by Merck Group (1 mentions) Tgf β, by Thermo Fisher Scientific (1 mentions) Interleukin 2 (il 2), by BD (1 mentions) Dnase 1, by Roche (1 mentions) Facsaria 3, by BD (1 mentions) Collagenase a, by Roche (1 mentions) Anti cd3 antibody, by BioXCell (1 mentions)

7 protocols using easysep mouse cd90.2 positive selection kit 2

Isolation of Tumor-Infiltrating T Cells

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Mice were inoculated with 5.0×10⁵ MB49 or MB49OVA cells subcutaneously at the right ventral abdomen or 1.0×10⁵ MB49 or MB49OVA cells orthotopically. T cells from MB49 or MB49OVA tumors were isolated using EasySep Mouse CD90.2 Positive Selection Kit II (Stem Cell Technologies) according to the manufacturer’s protocol. Isolated cells were plated in CM+IL-2 (100 IU, Prometheus).

Bunch B.L., Morse J., Asby S., Blauvelt J., Aydin A.M., Innamarato P., Hajiran A., Beatty M., Poch M, & Pilon-Thomas S. (2020). Systemic and intravesical adoptive cell therapy of tumor-reactive T cells can decrease bladder tumor growth in vivo. Journal for Immunotherapy of Cancer, 8(2), e001673.

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Isolated Tumor-Infiltrating Lymphocyte Activation

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TILs from mice with MC38 tumors were isolated after treatment with isotype or α41BB antibodies using EasySep Mouse CD90.2 Positive Selection Kit II (STEMCELL Technologies). TILs were cultured in round-bottom 96 well plates with immobilized anti-CD3 antibodies (145–2C11 BD Biosciences) at a concentration of 5μg/mL or with irradiated tumor cell lines. MC38 or irrelevant target B16 tumor cells were exposed to X-ray irradiation at a dose of 2×10⁴ rad and cultured with CD90.2⁺ TILs at a 1:10 (target:TIL) ratio for 48hrs. Supernatants were collected and IFN-gamma was measured by (Mouse IFN-gamma Quantikine ELISA Kit, R&D Systems).

Innamarato P., Asby S., Morse J., Mackay A., Hall M., Kidd S., Nagle L., Sarnaik A.A, & Pilon-Thomas S. (2020). Intratumoral activation of 41BB co-stimulatory signals enhances CD8 T cell expansion and modulates tumor-infiltrating myeloid cells. Journal of immunology (Baltimore, Md. : 1950), 205(10), 2893-2904.

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Isolation and Culture of ILC2s

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For in vitro experiments, all the work was carried out aseptically and single cell suspensions were obtained from lungs and bone marrow of untreated Rag2^−/− mice. Whole lung and bone marrow cells were cultured in complete RPMI with a cytokine cocktail (IL‐2, IL‐7, and IL‐33) with various reagents indicated in the figure legends at 37°C, 5% CO₂ for 3–4 days. IL‐5 and IL‐13 in the supernatants were analysed by ELISA. In some experiments, CD90.2⁺ cells were pre‐sorted from Rag2^−/− lung and bone marrow single cell suspensions using an EasySep™ Mouse CD90.2 Positive Selection Kit II (Stemcell Technologies) according to manufacturer's instructions. Live Lineage(CD11c/CD11b/NK1.1)⁻CD45⁺CD90.2⁺ST2⁺ ILC2s were sorted using a BD FACS Aria II and then cultured in complete RPMI containing 50 μM β‐mercaptoethanol with IL‐2, IL‐7, and IL‐33 with or without PGE₂ in a round bottom 96‐well plate at 37°C in 5% CO₂ for 3 days.

Robb C.T., Zhou Y., Felton J.M., Zhang B., Goepp M., Jheeta P., Smyth D.J., Duffin R., Vermeren S., Breyer R.M., Narumiya S., McSorley H.J., Maizels R.M., Schwarze J.K., Rossi A.G, & Yao C. (2022). Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses. Allergy, 78(3), 714-730.

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Allo-HCT Transplantation in BALB/c Mice

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Allo-HCT was performed as previously described [12 (link),13 (link),15 (link),25 (link),26 (link),27 (link),32 (link)]. Briefly, BALB/c mice were used as recipients and received 9 Gy total body irradiation on day −1, then were transplanted with 5 × 10⁶ T cell-depleted bone marrow (TCD-BM) cells from B6 (CD45.1+) mice along with 0.5 × 10⁶ splenic T cells from B6 (CD45.2+) mice on day 0. Mouse TCD-BM and splenic T cells were prepared using an EasySep mouse CD90.2 positive selection kit II and an EasySep mouse T cell isolation kit (STEMCELL Technologies, Cambridge, MA, USA), respectively.

Kim S., Ruminski P., Singh M., Staser K., Ashami K., Ritchey J., Lim S., DiPersio J.F, & Choi J. (2024). Novel JAK Inhibitors to Reduce Graft-Versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation in a Preclinical Mouse Model. Molecules, 29(8), 1801.

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Isolation and Culture of Naive CD4+ T Cells

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Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll density gradient centrifugation (Histopaque, SigmaAldrich) at 490g. Naïve CD4⁺ T cells were enriched from murine splenocytes (CD44^loCD62^hi) or human peripheral blood mononuclear cells (CD45RA⁺CD45RO⁻) (EasySep™ Mouse Naïve CD4⁺ T Cell Isolation Kit, EasySep™ Human Naïve CD4⁺ T Cell Isolation Kit, respectively, STEMCELL Technologies) and their purity checked by flow cytometry. Mouse cultures: 200,000 naïve CD4⁺ T cells were incubated with IL-2 (2.75 ng/ml, Peprotech), TGFβ (0.7 ng/ml, Peprotech) ± IFNβ (100 U/ml or 1,000 U/ml, PBL Biosciences) or IFNα2 (1000 U/ml) or IFNα4 (1000 U/ml and stimulated with either αCD3/αCD28 (15 μl/million cells, Gibco) (polyclonal), or 200,000 BALB/c APCs (EasySep™ Mouse CD90.2 Positive Selection Kit II, STEMCELL Technologies) cells previously incubated with anti-IFNAR1 antibody for 1 h (10ug/ml, Bio X Cell) (allostimulation). Human cultures: 200,000 naïve CD4⁺ T cells were, in certain cases previously incubated with anti-IFNAR1 antibody or isotype for 30 min (1:1000 dilution, Abcam), and then cultured with IL-2 (100 U/ml, BD Pharmingen), TGFβ (3 ng/ml, Peprotech) ± IFNβ (100 U/ml, or 1000 U/ml, PBL Biosciences) and stimulated with aCD3/aCD28 (15 μl/million cells, Gibco).

González F.F., McGinty M., Ningoo M., Anderson L., Cantarelli C., Angeletti A., Demir M., Llaudó I., Purroy C., Marjanovic N., Heja D., Sealfon S.C., Heeger P.S., Cravedi P, & Fribourg M. (2022). Interferon-β Acts Directly on T cells to Prolong Allograft Survival by Enhancing Regulatory T cell Induction through Foxp3 Acetylation. Immunity, 55(3), 459-474.e7.

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Isolation and Characterization of Tumor-Infiltrating T Cells

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Tumor tissues were excised and digested with 1 mg/mL collagenase A (Roche) and 0.5 mg/mL DNase I (Roche) at 37°C for 30 minutes and then passed through a 70-μm cell strainer to remove large pieces of undigested tumor fragments. CD90⁺ cells were isolated with EasySep Mouse CD90.2 Positive Selection Kit II (STEMCELL Technologies Inc.) according to the manufacturer’s instructions. CD90⁺ cells were then stained with surface marker antibodies. CD4⁺, PD-1^–CD8⁺, PD-1⁺TIM3^–CD8⁺, and PD-1⁺TIM3⁺CD8⁺ T cells were sorted by BD FACSAria III (BD Biosciences).

Ren Z., Zhang A., Sun Z., Liang Y., Ye J., Qiao J., Li B, & Fu Y.X. (2022). Selective delivery of low-affinity IL-2 to PD-1+ T cells rejuvenates antitumor immunity with reduced toxicity. The Journal of Clinical Investigation, 132(3), e153604.

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Regulatory T-cell Suppression Assay

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Treg suppression assays were carried out as described (25 (link)). Briefly, the assay was carried out in 96 well plates, with three purified/sorted cell populations in complete RPMI 1640 medium with 10% FBS: (i) irradiated (30Gy) T-cell-depleted splenocytes from WT mice (EasySep Mouse CD 90.2 positive selection kit II) (Stem cell technologies); (ii) CD4⁺CD25^- from WT mice; and (iii) CD4⁺CD25^HIGH cells from the TTPΔARE, or WT control mice, were sorted by FACS ARIAIII/Fusion cell sorter at the NEI flow cytometry core. Cell populations (i) and (ii) were added at 5 x 10⁵ in 50 μl/well, whereas cell populations (iii), from either WT or the TTPΔARE mice, were added in a series of double dilutions, in a volume of 50 μl/well. The T-cell populations were stimulated with 50 μl of anti-CD3 antibody (BioXcell) and incubated for 72 h, with 1 μCi of ³H thymidine being added for the last 8 h.

Xu B., Tang J., Lyu C., Wandu W.S., Stumpo D.J., Mattapallil M.J., Horai R., Gery I., Blackshear P.J, & Caspi R.R. (2021). Regulated Tristetraprolin Overexpression Dampens the Development and Pathogenesis of Experimental Autoimmune Uveitis. Frontiers in Immunology, 11, 583510.

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