Rhil 15

Manufactured by BioLegend

Sourced in United Kingdom, United States

RhIL-15 is a recombinant human interleukin-15 protein. Interleukin-15 is a cytokine that plays a role in the activation and proliferation of T cells and natural killer cells.

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Lab products found in correlation

Rhil 12, by BioLegend (4 mentions) Rhil 2, by BioLegend (3 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) Human ab serum, by Merck Group (2 mentions) Anti cd3, by Thermo Fisher Scientific (2 mentions) Aqua live dead viability dye, by Thermo Fisher Scientific (2 mentions) Anti cd3 percp cy5.5 ucht1, by BioLegend (2 mentions) Anti cd4 bv785 okt4, by BioLegend (2 mentions) Anti cd26 pe cy7 ba5b, by BioLegend (2 mentions) Rhil 7, by BioLegend (2 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Proleukin, by Novartis (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Easysep human nk cell enrichment kit, by STEMCELL (1 mentions) Nk macs medium, by Miltenyi Biotec (1 mentions) Non essential amino acids (neaa), by Corning (1 mentions) Sodium pyruvate, by Corning (1 mentions) Hepes, by Corning (1 mentions)

6 protocols using rhil 15

Isolation and Culture of Breast Tumor Lymphocytes

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For direct ex vivo isolation, fresh breast tumor or tissue was coarsely minced with scalpels and then dissociated using the MACS human tumor dissociation kit on a gentleMACS Dissociator as per manufacturer’s instructions (Miltenyi Biotec). Samples were washed twice with sterile RPMI-1640 and used immediately for downstream assays. Lymphocytes were also harvested using a “grid” explant system adapted from a protocol first described by Clark and colleagues (35 (link)). Briefly, fresh breast tumor or tissue was minced using scalpels and placed onto rat tail collagen (100μg/ml, BD Biosciences)-coated Cellfoam grids (Cytomatrix Pty Ltd). Each grid was placed into a separate well of a 24-well tissue culture plate and cultured in complete medium [Iscove’s Modified Dulbecco’s Medium (IMDM, Life Technologies), 10% heat-inactivated foetal bovine serum (FBS, Life Technologies), L-glutamine (292μg/ml, Life Technologies), penicillin (100units/ml, Life Technologies), streptomycin (100μg/ml, Life Technologies) and 2-mercaptoethanol (3.5μl/L, Life Technologies)] supplemented with recombinant human (rh) IL-2 (100 IU/ml, Proleukin, Novartis Pharmaceuticals UK Ltd), and rhIL-15 (10 ng/ml, BioLegend). The grids were maintained for 3 weeks in culture at 37°C/5% CO₂, and the lymphocytes harvested by washing the wells/grids with 0.01 mM HEPES/HBSS.

Wu Y., Kyle-Cezar F., Woolf R.T., Naceur-Lombardelli C., Owen J., Biswas D., Lorenc A., Vantourout P., Gazinska P., Grigoriadis A., Tutt A, & Hayday A. (2019). An innate-like Vδ1+ γδ T cell compartment in the human breast is associated with remission in Triple Negative Breast Cancer. Science translational medicine, 11(513), eaax9364.

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Expansion and Purification of Primary NK Cells

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Primary NK cells were isolated from at least 5 x 10⁶ cells of cryopreserved PBMCs using the EasySep Human NK cell Enrichment Kit according to the manufacturer’s instructions (STEMCELL Technologies, Canada). The purity of NK cells was > 95% of CD3^-CD56⁺ cells as determined by flow cytometry. Primary NK cells obtained from healthy donors (n =4) were cultured and expanded in NK MACs medium (Miltenyi Biotec, Bergisch-Gladbach, Germany) supplemented with 10% human AB serum (Sigma,), 1 mM Penicillin/Streptomycin (Sigma), 5 ng/ml of rhIL-2 and rhIL-15 (Biolegend, CA, USA) at 37°C with 5% CO₂ for 7-10 days. Expanded NK cells were used in the experimental validation of the NK cell memory assay.

Preechanukul A., Kronsteiner B., Saiprom N., Rochaikun K., Moonmueangsan B., Phunpang R., Ottiwet O., Kongphrai Y., Wapee S., Chotivanich K., Morakot C., Janon R., Dunachie S.J, & Chantratita N. (2023). Identification and function of a novel human memory-like NK cell population expressing CD160 in melioidosis. iScience, 26(8), 107234.

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MAIT Cell Cloning Protocol

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MAIT cell cloning was performed as described by Cansler et al. (22 ). Briefly, MR1 tetramer-positive T cells were sorted from cryopreserved bronchoalveolar lavage cells and rested overnight. Limiting dilution assay was performed to seed single cells in 96-well plates in RPMI media containing 10% human serum (HuS), 2% L-glutamine, 0.1% gentamycin and irradiated feeder cells (lymphoblastoid cell lines and peripheral blood mononuclear cells). Media was supplemented with recombinant human (rh)IL-2 (5 ng/mL), rhIL-7 (0.5 ng/mL), rhIL-12 (0.5 ng/mL), rhIL-15 (0.5 ng/mL) (BioLegend) and anti-CD3 (0.03 µg/mL) (eBiosciences). Plates were assessed weekly for growth with clones taking 2 – 3 weeks to grow. Clones were taken forward for subsequent analyses only from plates, which had growth in approximately less than 30% of the wells. Resulting clones were subjected to surface MR1 5-OP-RU tetramer and monoclonal antibody staining with 1:200 of Live/dead viability dye (Aqua, Life Technologies), 1:25 of anti-CD3 PerCP/Cy5.5 (UCHT1, BioLegend), 1:25 of anti-CD4 BV785 (OKT4, BioLegend), 1:50 of anti-CD8 (FITC, RPA-T8) and 1:25 of anti-CD26 PE-Cy7 (BA5b, BioLegend), as well as IFN-γ ELISPOT assay to confirm MAIT cell identify and clonal purity.

Khuzwayo S., Mthembu M., Meermeier E.W., Prakadan S.M., Kazer S.W., Bassett T., Nyamande K., Khan D.F., Maharaj P., Mitha M., Suleman M., Mhlane Z., Ramjit D., Karim F., Shalek A.K., Lewinsohn D.M., Ndung’u T, & Wong E.B. (2021). MR1-Restricted MAIT Cells From The Human Lung Mucosal Surface Have Distinct Phenotypic, Functional, and Transcriptomic Features That Are Preserved in HIV Infection. Frontiers in Immunology, 12, 631410.

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CD16 Ligation Assay for NK Cell Activation

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For the CD16 ligation assay, 0.5 × 10⁶ to 1 × 10⁶ PBMCs were stimulated in 96-well round-bottom plates in a 37°C incubator with 5% CO2. PBMCs were thawed and cultured for 12 to 14 hours with basal medium [RPMI 1640 containing 10% Human AB serum (Sigma-Aldrich), 10 mM Hepes (Corning), 1× nonessential amino acids (Corning), 1 mM sodium pyruvate (Corning), 1× penicillin (100 IU/ml)–streptomycin (100 μg/ml) solution (Gibco), 2 mM L-glutamine (Gibco)] and recombinant human IL-15 (rhIL-15) (1 ng/ml) (Miltenyi). Before stimulation, medium was changed to fresh medium containing indicated concentrations of anti-CD16 (BD Biosciences) antibody–conjugated MACS iBeads (Miltenyi). After 3 hours, cells were harvested for flow cytometric analysis as described above. For cytokine priming assays, replicates of 10⁶ PBMCs harvested from a healthy donor Leukopak (STEMCELL) were incubated overnight in basal medium containing rhIL-15 (1 ng/ml), rhIL-12 (10 ng/ml) (BioLegend), and rhIL-18 (100 ng/ml) (Gibco). For purified NK cell stimulation, NK cells were isolated from Leukopak PBMCs using the Human NK Cell Negative Selection Kit (STEMCELL) in a 96-well round-bottom plate using an EasyPlate magnet (STEMCELL) per the manufacturer’s instructions.

Mack M.R., Brestoff J.R., Berrien-Elliott M.M., Trier A.M., Yang T.L., McCullen M., Collins P.L., Niu H., Bodet N.D., Wagner J.A., Park E., Xu A.Z., Wang F., Chibnall R., Council M.L., Heffington C., Kreisel F., Margolis D.J., Sheinbein D., Lovato P., Vivier E., Cella M., Colonna M., Yokoyama W.M., Oltz E.M., Fehniger T.A, & Kim B.S. (2020). Blood natural killer cell deficiency reveals an immunotherapy strategy for atopic dermatitis. Science translational medicine, 12(532), eaay1005.

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NK cell Stimulation Assay

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Unprimed or primed NK-92 MI cells resuspended in 500μl of cMEM-α media were seeded at 5×10⁵ live cells/well into 24-well tissue culture plates. The cells were stimulated with 5 ng/ml of rhIL-12, rhIL-15 and rhIL-18 (Biolegend) alone or in combination for 18 h. Phorbol myristic acetate (PMA) (20 ng/ml; Sigma) plus ionomycin (1 μg/ml; Sigma) were used as positive control. Cell-free culture supernatant was collected by centrifugation at 1,000× g for 5 min and measured for IFN-γ production by Human IFN-γ ELISA Set (BD Biosciences) according to the manufacturer’s instructions. For in vitro re-stimulation, primary NK cells obtained from a clinical cohort were resuspended in R10 and then plated at 1.25×10⁵ live cells/well into 96-well round-bottom culture plate. The cells were incubated in the absence or presence of iBP (MOI 100) for 18 h. Unstimulated or stimulated NK cells were subsequently stimulated with 5 ng/ml of rhIL-12 and rhIL-18 (Biolegend CA, USA) for 18 h. NK cells were analysed for IFN-γ production using flow cytometry.

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Cloning and Characterizing MAIT Cells

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MAIT cell cloning was performed as described by Cansler et al. [20] . Briefly, MR1 tetramer-positive T cells were sorted from cryopreserved bronchoalveolar lavage cells and rested overnight. Limiting dilution assay was performed to seed single cells in 96-well plates in RPMI media containing 10% human serum (HuS), 2% L-glutamine, 0.1% gentamycin and irradiated feeder cells (lymphoblastoid cell lines and peripheral blood mononuclear cells). Media was supplemented with recombinant human (rh)IL-2 (5 ng/mL), rhIL-7 (0.5 ng/mL), rhIL-12 (0.5 ng/mL), rhIL-15 (0.5 ng/mL) (BioLegend) and anti-CD3 (0.03 µg/mL) (eBiosciences). Plates were assessed weekly for growth with clones taking 2 -3 weeks to grow. Clones were taken forward for subsequent analyses only from plates which had growth in approximately less than 30% of the wells. Resulting clones were subjected to surface MR1 5-OP-RU tetramer and monoclonal antibody staining with 1:200 of Live/dead viability dye (Aqua, Life Technologies), 1:25 of anti-CD3 PerCP/Cy5.5 (UCHT1, BioLegend), 1:25 of anti-CD4 BV785 (OKT4, BioLegend), 1:50 of anti-CD8 (FITC, RPA-T8) and 1:25 of anti-CD26 PE-Cy7 (BA5b, BioLegend), as well as IFN-γ ELISPOT assay to confirm MAIT cell identify and clonal purity.

Khuzwayo S., Mthembu M., Meermeier E.W., Prakadan S.M., Kazer S.W., Bassett T., Nyamande K., Khan D.F., Maharaj P., Mitha M., Suleman M., Mhlane Z., Ramjit D., Karim F., Shalek A.K., Lewinsohn D., Ndung’u T., & Wong E. (2020). MR1-restricted MAIT cells from the human lung mucosal surface have distinct phenotypic, functional, and transcriptomic features that are preserved in HIV infection.

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