Human embryonic kidney cell line 293 (HEK293) stably expressing human integrin αvβ3 (293 + αvβ3) (LaFrance et al., 2011 (link)) was cultured in Dulbecco’s modified Eagle medium plus Ham’s F12 nutrient mix (base medium; Gibco) with 5% heat-inactivated fetal bovine serum (FBS) (Gibco) supplemented with 400 μg ml−1 of G418 (Gibco). Human microvascular endothelial cells (HMEC-1) (Ades et al., 1992 (link)) were cultured in MCDB 131 (base medium; Gibco) with 15% heat-inactivated Hyclone FBS (Hyclone Laboratories), 1 μg ml−1 of hydrocortisone (Sigma-Aldrich), 10 ng ml−1 of epidermal growth factor (Gibco) and 25 mM HEPES (Gibco). The mouse lung epithelial cell line LA-4 was cultured in Kaighn’s modification of Ham’s F12 medium (F12K) (base medium; Gibco) with 10% heat-inactivated FBS (Gibco) (Stoner et al., 1975 (link)). All cell lines were cultured in the presence of additional L-glutamine (Gibco) to a final concentration of 2 mM and for general maintenance of cell lines, penicillin at a concentration of 100 U ml−1 and streptomycin to a final concentration of 100 μg ml−1 (Gibco). For bacterial infections, antibiotic containing medium was removed and cells were washed before being lifted with Trypsin-EDTA (Gibco) and suspended in antibiotic free medium prior to being plated in the absence of antibiotics. Cells were incubated at 37°C under 5% CO2.
Hyclone fbs
Manufactured by Cytiva
Sourced in United States
HyClone FBS is a high-quality fetal bovine serum (FBS) product offered by Cytiva. It is designed to provide essential nutrients and growth factors to support cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Mcdb131, by Thermo Fisher Scientific (2 mentions)
Ham s f 12 nutrient mix, by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Nunc six well plates, by Thermo Fisher Scientific (2 mentions)
Accell transfection media, by Horizon Discovery (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Interleukin 2 (il 2), by R&D Systems (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin 100, by Euroclone (1 mentions)
Cpg b odn2006, by Hycult Biotech (1 mentions)
Rpmi 1640, by Euroclone (1 mentions)
Roosternourish msc, by RoosterBio (1 mentions)
16 protocols using hyclone fbs
1
Culturing of Human and Murine Cell Lines
2
Polyclonal Stimulation of PBMCs
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As we published before [14 (link)], in order to induce antibody production from MBCs, we polyclonally stimulated PBMCs with CpG. Briefly, PBMCs were cultured in complete medium at a concentration of 1 × 106 cells/mL. Complete medium was prepared as follows: RPMI-1640 (Euroclone, Milan, Italy), 10% heat inactivated HyClone FBS (Hyclone Laboratories, Logan, UT, USA), 1mM L-Glutamine (GIBCO BRL, New York, NY, USA); 1× Penicillin/Streptomycin 100× (Euroclone, Milan, Italy), 1% sodium pyruvate (GIBCO BRL, New York, NY, USA). Cells were stimulated for 5 days with 0.35 µM TLR9 agonist CpG-B ODN2006 (Hycult Biotech, Uden, The Netherland) plus 20 ng/mL rhIL-21 (Milteny, Bergisch Gladbach, Germany) and 20 ng/mL rhIL-4 (Milteny, Bergisch Gladbach, Germany).
3
Culture of Mammalian Cell Lines
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Human embryonic kidney cell line 293 (HEK293) stably expressing human integrin αvβ3 (293 + αvβ3) (LaFrance et al., 2011 ) was cultured in Dulbecco's modified Eagle medium plus Ham's F12 nutrient mix (base medium; Gibco) with 5% heat‐inactivated fetal bovine serum (FBS) (Gibco) supplemented with 400 μg ml−1 of G418 (Gibco). Human microvascular endothelial cells (HMEC‐1) (Ades et al., 1992 ) were cultured in MCDB 131 (base medium; Gibco) with 15% heat‐inactivated Hyclone FBS (Hyclone Laboratories), 1 μg ml−1 of hydrocortisone (Sigma‐Aldrich), 10 ng ml−1 of epidermal growth factor (Gibco) and 25 mM HEPES (Gibco). The mouse lung epithelial cell line LA‐4 was cultured in Kaighn's modification of Ham's F12 medium (F12K) (base medium; Gibco) with 10% heat‐inactivated FBS (Gibco) (Stoner et al., 1975 ). All cell lines were cultured in the presence of additional L‐glutamine (Gibco) to a final concentration of 2 mM and for general maintenance of cell lines, penicillin at a concentration of 100 U ml−1 and streptomycin to a final concentration of 100 μg ml−1 (Gibco). For bacterial infections, antibiotic containing medium was removed and cells were washed before being lifted with Trypsin‐EDTA (Gibco) and suspended in antibiotic free medium prior to being plated in the absence of antibiotics. Cells were incubated at 37°C under 5% CO2.
4
Isolation and Expansion of Human MSCs
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Human mesenchymal stromal cells (MSCs) were isolated from single donor bone marrow (Lonza, Walkersville, MD, USA) based upon their adherence to tissue-culture treated flasks in standard conditions. MSCs were cultured in minimal essential media-α (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2.5 ng/mL rec. human FGF-2 (Waisman Biomanufacturing, Madison, WI, USA), 10% v/v Hyclone FBS (Cytiva, Marlborough, MA, USA) and 1% v/v antibiotic- antimycotic (Thermo Fisher Scientific) at 37°C/5% CO2.
Working Cell Bank (MSC-WCB, Passage: 2) and Cell Therapy Product (MSC-CTP, Passage: 3) stocks of MSCs were used for the entirety of this study. Once thawed, cells were seeded at a density 3,000–3,500 cells/cm2 and weaned to xeno-free conditions using RoosterNourish-MSC (Rooster Bio, Fredrick, MD, USA). After 4 days, MSCs were dissociated from flasks using TrypLE Express Enzyme (Thermo Fisher Scientific) and counted using an NC-202 automated cell counter (ChemoMetec, Allerod, Denmark). Cell suspensions were centrifuged at 1100 rpm for 5 min and resuspended for encapsulation.
Working Cell Bank (MSC-WCB, Passage: 2) and Cell Therapy Product (MSC-CTP, Passage: 3) stocks of MSCs were used for the entirety of this study. Once thawed, cells were seeded at a density 3,000–3,500 cells/cm2 and weaned to xeno-free conditions using RoosterNourish-MSC (Rooster Bio, Fredrick, MD, USA). After 4 days, MSCs were dissociated from flasks using TrypLE Express Enzyme (Thermo Fisher Scientific) and counted using an NC-202 automated cell counter (ChemoMetec, Allerod, Denmark). Cell suspensions were centrifuged at 1100 rpm for 5 min and resuspended for encapsulation.
5
Isolation and Cryopreservation of Human Airway Epithelial Cells
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Human AECs were isolated from discarded bronchial segments from donor lungs that were collected for transplant according to the protocol approved by University of Arizona’s Institutional Review Board (under PI, Dr. Polverino). Upon arrival in the lab, the bronchus was rinsed with RPMI1640 and submerged in 1 mg/ml protease (Sigma-Aldrich, St. Louis, MO) in Ham’s F12 for 1 h at 37°C. After neutralizing with HyClone FBS (Cytiva, Marlborough, MA), AECs were gently scrapped out and incubated in Gibco Versene (Thermo Fisher Scientific, Waltham, MA). AECs were washed and seeded on a 100 mm collagen and fibronectin-coated tissue culture dish in PneumaCult EX Plus supplemented with 10 µM ROCK inhibitor (APExBIO, Houston, TX) until 90% confluency. Cells were collected using trypsin and preserved in PneumaCult EX Plus (STEMCELL Technologies, Vancouver, BC) containing 30% FBS and 10% DMSO at 106 cells/ml and stored at −80°C.
6
Gene Silencing with siRNA Depletion
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Gene silencing with siRNA was performed as described (23 (link), 30 (link), 49 (link)). In brief, to deplete α1B/D-ARs from the cell surface, cells were incubated in Nunc six-well plates (Thermo Fisher Scientific) for 3 d in Accell transfection media (GE Dharmacon) with α1B-AR, α1D-AR, or NT (negative control) siRNA (GE Dharmacon) at a concentration of 1 µM. On day 3, cells were centrifuged at 300 × g for 5 min and resuspended in RPMI 1640 (Sigma) supplemented with 10% HyClone FBS from Cytiva (Marlborough, MA), 100 μg/mL penicillin from Invitrogen (Waltham, MA), and 100 μg/mL streptomycin (Invitrogen). Cells were utilized on day 4 for experimentation.
7
Evaluating GPC-3.CAR and GPC-3.CAR/sIL-15 Vδ1 T Cell Cytotoxicity
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GPC-3.CAR and GPC-3.CAR/sIL-15 Vδ1 T cells were co-cultured for 24 hours with HepG2 or PLC/PRF/5 cells at a 2:1 E:T ratio in RPMI 1640 medium (Gibco) supplemented with 10% HyClone FBS (Cytiva). MILLIPLEX Immunology Multiplex Assay Panel (HCYTMAG-60K-PX29, Millipore, Burlington, Massachusetts) was used according to manufacturer’s protocol to measure cytokine and chemokine levels in culture supernatants on the FLEXMAP three-dimensional (3D) Instrument (Millipore).
8
Characterization of TNBC Cell Lines
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The TNBC cell line 4T1 (CRL-2539) was obtained from ATCC. The D2F2/E2 cell line was a kind gift from Dr. Wei-Zen Wei from Wayne State University. D2F2/E2 cells were generated by transfecting D2F2 cells with full length human ErbB-2 (HER2) [19 (link)]. Cell lines were verified using their expression profile of surface markers and morphology. Mycoplasma testing was performed routinely as a precautionary quality check. These cell lines were cultured in complete DMEM with 10% Hyclone FBS (Cytiva, Marlborough, MA, USA), 4500 mg/L glucose (MilliporeSigma, Burlington, MA, USA), Gibco Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), and Penicillin-Streptomycin (MilliporeSigma, Burlington, MA, USA). Selection medium for D2F2/E2 cells contained 800 μg/mL G418 (MilliporeSigma, Burlington, MA, USA).
9
Isolation and Expansion of Vδ1 T Cells
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PBMCs were isolated from leukapheresis material collected from single, eligible, cytomegalovirus-negative, healthy donors using red blood cell (RBC) lysis or a Ficoll-Paque gradient prior to cryopreservation (STEMCELL Technologies, Cambridge, Massachusetts). PBMCs were subsequently thawed and plated onto immobilized agonistic anti-Vδ1 antibody (Adicet Therapeutics). Antibody-activated PBMCs were transduced with the γ-retroviral CAR construct in combination with RetroNectin (Takara, Mountain View, California). Following transduction, cells were expanded in X-VIVO 15 medium (Lonza, Walkersville, Maryland) containing 10% HyClone FBS (Cytiva) and 100 IU/mL human IL-2 (Peprotech, Cranbury, New Jersey). Following expansion, remaining αβ T cells were labeled with biotinylated anti-TCRαβ antibody and anti-biotin immunomagnetic beads (Miltenyi, Auburn, California) and depleted using the autoMACS Pro (Miltenyi) prior to filling into vials and cryopreservation.
10
Stimulation and Culture of Peripheral T-Cells
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Pan-CD3+ peripheral T-cells were purchased from HemaCare (PB03C-1). Cells were thawed in a 37 °C water bath before adding 10x volume of 37 °C HBSS + 10% FBS dropwise. T-cells were then centrifuged for 10 min at 300×g and resuspended in 37 °C IMDM + 10% Hyclone FBS (Cytiva) with 10 ng/ml IL-2 (R&D Systems) at 1–2 M cells/ml and cultured for 48 h prior to nonspecific TCR stimulation and cytokine secretion experiments.
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