ExCellerate™ Human NK Cell Expansion Media, Xeno-Free
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ExCellerate™ Human NK Cell Expansion Media, Xeno-Free Xeno-Free Media for Human Natural Killer Cell Culture Catalog Number: CCM032 Volume: 500 mL PRODUCT DESCRIPTION Natural Killer (NK) cells play an important role in both the adaptive and innate immune responses that control infection, autoimmunity, and tumor immunosurveillance (1). Human NK cells are phenotypically characterized by the expression of CD56 and the absence of CD3 (2). NK cells produce immunoregulatory cytokines, including interferon-γ (IFN-γ), tumor necrosis factor-β (TNF-β), tumor necrosis factor-α (TNF-α), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-10, and IL-13. Due to their intrinsic and non-specific anti-tumor activity, human NK cells have been employed as anti-cancer therapies (3-9). Furthermore, target-specific engineered NK cells that express Chimeric Antigen Receptors (CARs) enable greater precision in the treatment of malignancies (10-11). To facilitate discovery and preclinical research for NK cell immunoregulation and therapy, robust xeno-free and feeder-free platforms for ex vivo expansion and maintenance of NK cells are required. Traditional methods for NK cell expansion employ serum-containing media or artificial antigen-presenting K562 feeder cells. Serum and feeder cells introduce uncontrolled variables into defined culture that can confound NK expansion and killing activity. ExCellerate Human NK Cell Expansion Media, Xeno-free, is specially formulated and optimized for the ex vivo feederfree culture of human NK lymphocytes for research application. Unlike traditional serum-containing NK media, this xeno-free product provides a stable and reproducible cell culture environment for the expansion of NK cells under serum-free and feeder-free conditions. This product does not contain antibiotics or any non-human animal-derived components. INTENDED USE ExCellerate Human NK Cell Expansion Media, Xeno-Free, is a versatile media that can be used with a variety of cell activation methods and cytokines to culture and expand human NK cells ex vivo. The activation and cytokine/growth factor combination used with this media should be optimized by application or experimental protocol. STABILITY & STORAGE Upon receipt, this media should be stored at ≤ -20 °C. Upon thawing, store at 2-8 °C for up to 28 days. PRECAUTIONS The human origin-derived components used in this product have been derived from human plasma, which has been tested at the donor level and found to be negative for antibodies to HIV-1/2, hepatitis B surface antigen (HBsAg), and hepatitis C virus (HCV). All other components are animal-free. The media should be handled as if potentially infectious. Safe laboratory procedures should be followed and protective clothing should be worn when handling this media. The acute and chronic effects of over-exposure to this media are unknown. When handling bio-hazardous materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn. LIMITATIONS • FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. • The safety and efficacy of this product in diagnostic or other clinical uses has not been established. • This reagent should not be used beyond the expiration date indicated on the label. • Results may vary due to variations among primary NK lymphocyte populations derived from different donors. • The most recent version of the End User Terms of Use of Product may be found at: RnDSystems.com/Legal-information. 727047.2 03/23 Global bio-techne.com info@bio-techne.com TEL +1 612 379 2956 USA TEL 800 343 7475 Europe | Middle East | Africa TEL +44 (0)1235 529449 China TEL +86 (21) 52380373 For research use only. Trademarks and registered trademarks are the property of their

THE FOLLOWING MATERIALS ARE REQUIRED FOR THE PROCEDURES BELOW • Recombinant Human IL-2 (R&D Systems®, Catalog # 202-IL) • Recombinant Human IL-12 (R&D Systems, Catalog # 219-IL) • Recombinant Human IL-18 (R&D Systems, Catalog # 9124-IL) • Recombinant Human IL-21 (R&D Systems, Catalog # 8879-IL) • Sterile Phosphate Buffered Saline (PBS) • Pipettes and pipette tips • 15 mL and 50 mL Polypropylene Centrifuge Tubes • 24-well tissue culture plate • 25 cm2 tissue culture flask (T25) • 75 cm2 tissue culture flask (T75) • 37 °C, 5% CO2 incubator • Inverted microscope • Flow cytometer • Cell...

PROTOCOL USING PLATE-BOUND ANTIBODY Day 0 1. Pre-warm the required amount of 1X Complete ExCellerate™ Human NK Cell Expansion Media to room temperature. 2. Pre-coat a 24-well tissue culture plate with 10 μg/mL of Human NKp46/NCR1 Antibody (R&D Systems®, Catalog # MAB1850) in 1X PBS (250 µL per well to ensure well is completely covered). 3. Incubate plate for 1-3 hours at 37 °C. 4. Wash plate with 1 mL/well of sterile 1X PBS. 5. Isolate PBMCs or CD3+-depleted PBMCs using desired protocol. Determine the concentration of (NK cells/mL) in the starting population using flow cytometry. 6. Dilute...

Figure 1. Flow Cytometry Analysis of Expanded Human NK Cells. Human PBMCs were expanded in vitro for 14 days. NK Cells were collected on days 0, 8, 11, and 14 of expansion, and stained with Human CD3 Fluorescent-conjugated antibody (R&D Systems®, Catalog # FAB100F) and Human NCAM-1/CD56APC-conjugate antibody, and analyzed by flow cytometry. Compared to unexpanded PBMCs on Day 0, Days 8, 11, and 14 show an increased number of CD3- CD56+ NK Cells. Flow quadrants were set based on isotope-stained samples. % of Total Viable Cells Figure 2. NK Cell Expansion and Purity Following Expansion. Human...