Karpas 299 (ALK-positive anaplastic large cell lymphoma, a PTCL subtype) and HuT78 (CTCL) cell lines were obtained from ATCC, Gaithersburg, MD, USA. Mac-1 (developed by M.E.K.) was derived from circulating CTCL cells from a patient with multiple T-cell neoplasms [34 (link)]. Karpas 299 and Mac-1 were maintained in RPMI 1640 (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Clontech Laboratories, Inc, Mountain View, CA, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). HuT78 was maintained in Iscove's Modified Dulbecco's Medium (Gibco, Grand Island, NY, USA) containing 15% FBS and 1% penicillin/streptomycin. All cell lines were electroporated with 300V for 10 msec in antibiotic-free medium using the ECM 830 electroporation system (BTX Harvard Apparatus).
Karpas 299
Manufactured by American Type Culture Collection
Sourced in United States, Germany
The Karpas 299 is a laboratory equipment designed for the cultivation and maintenance of cell cultures. It provides a controlled environment for the growth and observation of various cell types, enabling researchers to study their characteristics and behaviors.
Automatically generated - may contain errors
Lab products found in correlation
Sup m2, by American Type Culture Collection (5 mentions)
Pen strep, by Thermo Fisher Scientific (5 mentions)
Stattic, by Merck Group (3 mentions)
Sr 786, by American Type Culture Collection (3 mentions)
Molt 4, by American Type Culture Collection (3 mentions)
Ccrf cem, by American Type Culture Collection (3 mentions)
Nk 92, by American Type Culture Collection (3 mentions)
Karpas 299, by Thermo Fisher Scientific (3 mentions)
Ccrf cem, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
U0126, by Cell Signaling Technology (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Jurkat, by American Type Culture Collection (2 mentions)
Su dhl 1, by American Type Culture Collection (2 mentions)
Hut78, by American Type Culture Collection (2 mentions)
Su dhl 1, by Leibniz Institute DSMZ (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ecm830 electroporation system, by Harvard Apparatus (1 mentions)
17 protocols using karpas 299
1
Cell Line Cultivation and Electroporation
2
Experimental Protocols for ALK+ ALCL Cell Lines
3
Myeloma Cell Lines and Patient Samples
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Two human MM cell lines, U266 and RPMI8226 cells were obtained from Dr. Linda Pilarski. Karpas 299 and SupM2 cells were purchased from ATCC. All cell lines were grown in RPMI1640 medium supplied with 10% FBS with 1% streptomycin and penicillin except U266 cells, which were grown in RPMI1640 medium supplied with 15% FBS. Ficoll-Paque isolated bone marrow mononuclear cells from two MM patients and reconstituted bortezomib in sterile water (1 mg/mL) were obtained from Cross Cancer Institute, University of Alberta. Both patients #1 and #2 contained 10–20% monoclonal plasma cells according to their biopsy section. Stattic (Sigma, Oakville, ON, Canada) powder was dissolved in DMSO into 1 mg/mL solution. All procedures of patient sample handling were approved by Human Research Ethics Board, University of Alberta (#Pro00058140). Animal procedures for this study were approved by Animal Care and Use Committee, University of Alberta (#Pro00000282).
4
Culturing Human Leukemia and NK Cells
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Human leukemia cells were obtained from residual samples on a protocol approved by the Institutional Review Board of Stony Brook University. Cord blood cells were also obtained under protocol from donors at Stony Brook University Hospital. Written, informed consent was obtained from all donors. KARPAS-299, HL-60, CCRF-CEM, MOLT4 and NK-92 cell lines were obtained from ATCC (Manassas, VA). NK-92 cells were cultured in filtered NK cell media, defined as alpha-MEM without ribonucleosides and deoxyribonucleosides with 2mM L-glutamine, 1.5 g/L sodium bicarbonate, 12.5% heat-inactivated horse serum, 12.5% heat-inactivated FBS, 1X Pen/Strep, 0.2% inositol, 0.02% folic acid, and 50 uM beta-mercaptoethanol, supplemented with IL-2 (300 IU/mL), unless otherwise specified. KARPAS-299, CCRF-CEM, and MOLT4 cell lines were cultured in RPMI, 10% FBS, 1x Pen/Strep (Gibco, Waltham, MA, USA). HL-60 cells were cultured in IMDM, 10% FBS, 1x Pen/Strep (Gibco, Waltham, MA, USA).
5
ALCL Cell Lines Maintenance and ΔNp63α Expression
6
Culturing Human Immune Cell Lines
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Human primary tumor samples were obtained from residual bone-marrow aspirate samples after final diagnosis was made according to compliance protocols. KARPAS 299, CCRF-CEM, Jurkat, MOLT-4, JeKo and NK-92 cell lines were obtained from ATCC (Manassas, VA). NK-92 cells were cultured in NK-92 cell media (defined as alpha-MEM without ribonucleosides and deoxyribonucleosides with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 12.5% heat-inactivated horse serum, 12.5% heat-inactivated FBS, 1 × Pen/Strep, 0.2% inositol, 0.02% folic acid and 50 uM beta-mercaptoethanol) supplemented with IL-2 (300 IU/ml) and fresh media every 2 days to a maintenance cell density of 0.3–1 × 106 cells/ml. NK-92 cells were then maintained for up to 3 months for in vitro and in vivo experiments. KARPAS 299, CCRF-CEM and Jurkat cell lines were cultured in RPMI, 10% FBS, 1 × Pen/Strep (Gibco, Waltham, MA, USA).
7
Experimental Protocols for ALK+ ALCL Cell Lines
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Three ALK+ ALCL cell lines were used in this study: Karpas 299 (a gift of Dr. M. Kadin, Beth Israel-Deaconess Medical Center, Boston, MA, USA), SR-786, and SUP-M2 (both from ATCC, Rockville, MD, USA). The T-cell acute lymphoblastic leukemia (T-ALL) cell line Jurkat was used as a negative control because of the absence of AP-1 activity. 293T is a cell line derived from human embryonic kidney (HEK) cells (ATCC). All cell lines were grown in Roswell Park Memorial Institute (RPMI)-1640 medium (Life Technologies, Grand Island, NY, USA) as described previously12 (link), supplemented with 10% fetal calf serum, and incubated at 37°C in a humidified atmosphere containing 5% CO2.
The JUNB expression vector pcDNA3.1/V5-His Topo was a gift from Dr. Anupam Agarwal (University of Alabama at Birmingham, Birmingham, AL, USA). The AKT1 luciferase promoter (−4293/+1888/Luc) was a gift from Dr. J. Q. Cheng (University of South Florida College of Medicine, Tampa, FL, USA). STAT 3 inhibitory compound Stattic (Sigma, St. Louis, MO, USA), a small-molecule inhibitor of STAT3 activation and dimerization, and the specific MEK1/2 inhibitor U0126 (Cell Signaling Technology, Beverly, MA, USA) were used.
The JUNB expression vector pcDNA3.1/V5-His Topo was a gift from Dr. Anupam Agarwal (University of Alabama at Birmingham, Birmingham, AL, USA). The AKT1 luciferase promoter (−4293/+1888/Luc) was a gift from Dr. J. Q. Cheng (University of South Florida College of Medicine, Tampa, FL, USA). STAT 3 inhibitory compound Stattic (Sigma, St. Louis, MO, USA), a small-molecule inhibitor of STAT3 activation and dimerization, and the specific MEK1/2 inhibitor U0126 (Cell Signaling Technology, Beverly, MA, USA) were used.
8
Cytotoxic Drug Combination Protocol
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4-Hydroperoxy-cyclophosphamide (4-HC, the active metabolite of cyclophosphamide), vincristine and doxorubicin, which compose the CHO treatment (2 µM 4-HC, 20 nM doxorubicin; 0.5 nM vincristine), were purchased from SIGMA-Aldrich (St. Louis, MO, USA). All other drugs were from Selleck Chem (Houston, TX, USA). For in vitro analyses, drugs were dissolved in DMSO; for in vivo studies, crizotinib and trametinib were prepared fresh in 0.5% carboxymethylcellulose/0.1% Tween 80; all the other compounds were dissolved in PBS. Primary antibodies (Table S1 ) were diluted 1:1000 in 5% BSA and incubated overnight. Anti-mouse and anti-rabbit secondary antibodies (Bio-Rad, Hercules, CA, USA) were used 1:2000 in 5% BSA. ALK+ ALCL cell lines Karpas-299, SUP-M2 and SU-DHL-1, and ALK-negative myelomonocytic leukemia U937 cells were purchased from ATCC. Cells were cultured in RPMI-1640 medium (Euroclone, Siziano, Italy) supplemented with 10% OptiClone fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin G, 80 μg/mL gentamicin and 20 mM HEPES, in a humidified atmosphere at 37 °C and 5% CO2.
9
ALCL Cell Lines Viability Assay
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ALCL cell lines Karpas299, SUDHL1, JB6, SR-786, SUP-M2, FE-PD, MAC1, and MAC2A were obtained from ATCC. All cell lines were cultured in RPMI-1640 supplemented with 10% FBS and 100 IU/mL penicillin. Cells were tested for mycoplasma every 3 months with the Mycoalert kit (Promega), and identity was confirmed by STR profiling (DFCI molecular diagnostics laboratory). Cell proliferation assays were performed by seeding 5000 cells per well in 96-well plates containing DMSO, crizotinib, alectinib, iberidomide, Stattic, or STAT3-IN-3 (MedChemExpress, LLC). Cell viability was assayed with CellTiter-Glo according to the manufacturer’s protocol (Promega).
10
Cell Line Validation Protocols
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Cell lines L540 (DSMZ, ACC72; RRID:CVCL_1362; human female), KARPAS 299 (DSMZ, ACC31, RRID:CVCL_1324; human male), HDLM-2 (DSMZ, ACC17; RRID:CVCL_0009; human male), and A20 (ATCC, TIB-208; RRID:CVCL_1940; mouse) were originally purchased from the vendors listed and since maintained by the Seagen cell bank (2016–2023). For each study, cell lines were requested from the cell bank and aliquots were thawed and cultured for periods between 1 and 3 weeks. Cell bank cell lines are routinely checked for contamination, including Mycoplasma, and cell line identity authentication by an STR-based DNA profiling and multiplex PCR, CellCheck 16–Human, or CellCheck 19 Mouse Plus, IDEXX Laboratories, Inc. Following any cell line modifications or cloning, expanded lines were resubmitted for pathogen burden and cell line identity validation before experimental use.
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