The T24 and J82 cell lines were purchased from the American Type Culture Collection (Rockville, MD) in December of 2008. After cells were received, the cell lines were frozen in liquid N2 after the first 3 passages with 50 ampules of cell stock. After an ampule was thawed, the cells were used for the designed experiments within 15 passages. They were grown in DMEM containing penicillin and streptomycin, supplemented with 10% fetal bovine serum (FBS). The normal immortalized non-transformed SV-HUC bladder urothelial cell line was purchased from American Type Culture Collection and grown in F-12k, supplemented with 10% FBS. In March 2012, the T-lymphocytic cell line HH (CD4+) was acquired from the American Type Culture Collection (ATCC# CRL-2105; CRL-1552) from ATCC and have been kept as 50 ampules of frozen cell stocks. HH cells were maintained in 10% RPMI (Invitrogen #A10491, Grand Island, NY) with 1% Pen/Strep and 10% FBS. After receipt from ATCC, all cell lines were used within 15 passages and were not re-authenticated by us. All cells were cultured in a 5% (v/v) CO2 humidified incubator at 37°C.
Crl 2105
Manufactured by American Type Culture Collection
Sourced in United States
The CRL-2105 is a cell culture vessel designed for the cultivation and maintenance of cells. It features a cell growth surface area of 25 cm^2 and is made of tissue culture-treated polystyrene. The vessel is suitable for a variety of cell types and provides a controlled environment for cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
Hh cd4, by American Type Culture Collection (1 mentions)
Jurkat clone e6 1, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by American Type Culture Collection (1 mentions)
Htb 176, by American Type Culture Collection (1 mentions)
Ser pbmc 200, by ZenBio (1 mentions)
Recombinant il32γ, by R&D Systems (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Wst 1 assay, by Roche (1 mentions)
9 protocols using crl 2105
1
Cell Line Sourcing and Maintenance
2
CRISPR/Cas9 Editing of HH T Cell Line
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HH cutaneous T cell lines (ATCC: CRL-2105), Jurkat E6–1 (ATCC:
TIB-152), and Daudi (ATCC: CCL-213, provided by Dr. Michael Brenner), were
cultured in complete RPMI as previously described. The HH cell line was chosen
for CRISPR/Cas9 experiments because it is a T cell line that expressed HLA class
II and has a transcriptome similar to the 72-hour time point in our dataset, at
which the largest effect of the Late-Spike haplotype occurs
(Supplementary Fig.
13 ).
TIB-152), and Daudi (ATCC: CCL-213, provided by Dr. Michael Brenner), were
cultured in complete RPMI as previously described. The HH cell line was chosen
for CRISPR/Cas9 experiments because it is a T cell line that expressed HLA class
II and has a transcriptome similar to the 72-hour time point in our dataset, at
which the largest effect of the Late-Spike haplotype occurs
(
13
3
CTCL Cell Lines for Research
4
Preparation and Characterization of Lymphoma Cell Lines
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Human lymphoma (HH, CRL-2105) cell line was from the ATCC. The human Hodgkin lymphoma (L-428, ACC 197) cell line was from the DSMZ. Human ataxia-telangiectasia mutated gene B lymphocyte cell line (AT, GM05126) was from the Coriell Institute. All cell lines were authenticated by the vendor, were maintained in a humidified incubator (37 °C, 5% CO2 and 95% air), and used at less than 10 passages from origin. Cells were propagated in DMEM & RPMI 1640 medium, respectively, supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2. Cells were cultured to the desired density (1 × 107 mL) and pelleted by centrifugation at 100× g at room temperature (RT) for 3 min. Pellets were washed with cold DPBS followed by 1× protease and phosphatase inhibitor solution in cold DPBS, and pelleted by centrifugation at 16,000× g at 4 °C for 20 min. Cell pellets were then lysed in RIPA lysis buffer supplemented with a protease and phosphatase inhibitor cocktail. Debris was removed by centrifugation at 15,000× g at 4 °C for 30 min, and the cleared lysate was stored at 4 °C.
5
Culturing Leukemic T Cell Lines
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HH cells (CRL-2105) and H9 cells (HTB-176) were obtained from ATCC (USA). According to ATCC, HH is a mature T cell line derived in 1986 from peripheral blood of a patient with aggressive cutaneous T cell leukemia/lymphoma while H9 cell line is a clonal derivative of the Hut 78 cell line and the H9 clone was selected for permissiveness for HIV-1 replication. Both the cell lines were cultured in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate (ATCC, USA). Cells were cultured in incubators with 5% CO2.
6
Bioactivity Validation of Compounds 3 and 7
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Example 19
To confirm bioactivity of 3 and 7, experiments were performed with the HH cell line, a mature T cell line derived from peripheral blood of a patient with aggressive cutaneous T cell leukemia/lymphoma (ATCC® CRL-2105™) which been demonstrated to only express the IL-2Rβ/γ. One of the earliest events in cytokine mediated activation of lymphocytes such as CD8+ T cells and NK cells is Janus Associated Kinase mediated phosphorylation and activation of Signal transducer and activator of transcription (pSTAT5). Thus, pSTAT5 was used to measure biological activity of 3 and 7 alongside 12. 3 demonstrated clear bioactivity in IL-2Rβ/γ expressing HH cells (EC50: 773 ng/ml) that was approximately 3.5 fold lower than 12 (EC50: 233 ng/ml). Additionally, 7 induced bioactivity (EC50: 756 ng/ml) very similar to 3, demonstrating that 7 retains bioactivity after being released from prodrug 5 even after accelerated (stress) conditions.
7
CTCL Cell Lines and PBMC Protocol
8
CRISPR/Cas9 Editing of HH T Cell Line
9
Characterization of MyLa and HH Cell Lines
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