Platinum-E (Plat-E) Retroviral Packaging cells (Cell Biolabs, Inc., Cat# RV-101) were provided by the Bluestone and Cyster Labs and cultured per the manufacturer’s instructions. Human embryonic kidney 293 cells (HEK293) were stored in the Fang lab and were cultured in DMEM containing 10% FBS. EG7 lymphoma, MC38 colon cancer, LLC1-OVA lung carcinoma and B16-SIY melanoma cell lines were provided by Dr. Bin Zhang and used for tumor models as previously reported48 . Cell lines were not genetically authenticated. HEK293 and cancer cell lines were tested for mycoplasma using LookOut Mycoplasma PCR detection kit (Sigma, Cat# MP0035–1KT). Myc-Usp22, Myc-Usp22(C185A), FLAG-Foxp3 and HA-ubiquitin expression plasmids and their tagged vectors were constructed and stored in the Fang lab. Antibodies used for Western blots, Co-IPs and flow cytometry are listed in Supplementary Table 4 . PMA (phorbol 12-myristate 13-acetate), ionomycin, and cycloheximide (CHX) were purchased from Sigma. Monesin was from eBioscience.
Platinum e plat e retroviral packaging cells
Manufactured by Cell Biolabs
Sourced in United States
The Plat-E (Platinum-E) retroviral packaging cells are a high-efficiency cell line used for the production of recombinant retroviruses. They are designed to provide a stable and reliable system for the generation of retroviral particles.
Automatically generated - may contain errors
Lab products found in correlation
Monesin, by Thermo Fisher Scientific (2 mentions)
Lookout mycoplasma pcr detection kit, by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Macs cell separation, by Miltenyi Biotec (1 mentions)
T cell enrichment column, by R&D Systems (1 mentions)
Easysep magnetic separation system, by STEMCELL (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Peg it virus concentration reagent, by System Biosciences (1 mentions)
Transporter 5 transfection reagent, by Polysciences (1 mentions)
El4 cells, by American Type Culture Collection (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Acrodisc filter, by Pall Corporation (1 mentions)
X tremegene 9 dna transfection reagent, by Roche (1 mentions)
X tremegene 9, by Roche (1 mentions)
7 protocols using platinum e plat e retroviral packaging cells
1
Culturing and Characterizing Cell Lines for Tumor Models
2
Culturing and Characterizing Cell Lines for Tumor Models
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Platinum-E (Plat-E) Retroviral Packaging cells (Cell Biolabs, Inc., Cat# RV-101) were provided by the Bluestone and Cyster Labs and cultured per the manufacturer’s instructions. Human embryonic kidney 293 cells (HEK293) were stored in the Fang lab and were cultured in DMEM containing 10% FBS. EG7 lymphoma, MC38 colon cancer, LLC1-OVA lung carcinoma and B16-SIY melanoma cell lines were provided by Dr. Bin Zhang and used for tumor models as previously reported48 . Cell lines were not genetically authenticated. HEK293 and cancer cell lines were tested for mycoplasma using LookOut Mycoplasma PCR detection kit (Sigma, Cat# MP0035–1KT). Myc-Usp22, Myc-Usp22(C185A), FLAG-Foxp3 and HA-ubiquitin expression plasmids and their tagged vectors were constructed and stored in the Fang lab. Antibodies used for Western blots, Co-IPs and flow cytometry are listed in Supplementary Table 4 . PMA (phorbol 12-myristate 13-acetate), ionomycin, and cycloheximide (CHX) were purchased from Sigma. Monesin was from eBioscience.
3
Isolation and Cultivation of Diverse Immune Cells
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Jurkat T cells (TIB-152), HEK293T (CRL-1573), Raji B cells (CCL-86; all from ATCC), and Platinum-E (Plat-E) retroviral packaging cells (Cell Biolabs, San Diego, CA, USA) were maintained in RPMI-1640 or Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen). Naive CD3+ T cells were purified from the mouse spleen and lymph nodes by negative selection using a T cell enrichment column (R&D Systems). To generate mouse T cell blasts, CD3+ T cells were incubated in 2 µg/mL anti-CD3/28-coated culture plates with 100 U/mL rIL-2 for 48 h and cultured for a further 5 days with 100 U/mL rIL-2. Mouse splenocytes were dispersed and purified into CD4+, CD8+, and CD19+ populations using the EasySep magnetic separation system (Stemcell Technologies, Vancouver, Canada) or MACS cell separation (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of each population was confirmed as >95% by flow cytometry. To establish B cell blasts, CD19+ cells from C57BL/6 wild-type mice were activated with LPS (10 μg/mL) for three days in complete RPMI. Bone marrow was flushed from femur and tibia bones, and bone-marrow-derived DCs (BMDCs) were grown with the addition of 20 ng/mL granulocyte macrophage-colony stimulating factor for five days.
4
PRMT5 Overexpression in CD4 T Cells
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pMRX- IRES-GFP retroviral vector, which contains a green fluorescent protein (GFP) reporter, was a kind gift from Dr. Leonid Pobezinsky (University of Massachusetts Amherst). The PRMT5 construct was cloned into the pMRX- IRES-GFP vector by GeneScript company (Piscataway, NJ). Empty pMRX-IRES-GFP vector was used as a control. Retroviral supernatants were produced by transfecting Platinum-E (Plat-E) retroviral packaging cells (Cell Biolabs, Inc, San Diego, CA) using Transporter 5 transfection reagent (Polysciences, Warrington, PA). Retroviral supernatants were concentrated with PEG-it™ virus concentration reagent (System Biosciences, Palo Alto, CA) prior to transduction. CD4 T cells were retrovirally transduced 24 h after activation with 10x concentrated retrovirus supernatants by spin-infection (660 × g for 90 min at 37°C) in the presence of polybrene (4 μg/ml). Transduction media was replaced with Th1-like iTreg polarizing media 4 h after spin-infection. Transduced cells were analyzed by flow cytometry and qRT-PCR.
5
Cell Culture Protocols for HEK293T, Plat-E, and EL4 Cells
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HEK293T cells (Cat#CRL-3216, ATCC, VA, USA), were grown in DMEM supplemented with 1% l -glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 55 μM 2-mercaptoethanol, and 10% FBS (10% DMEM). Platinum-E (Plat-E) retroviral packaging cells (Cat#RV-101, Cell Biolabs, CA, USA) were grown in 10% DMEM. EL4 cells (Cat# TIB-39, ATCC, VA, USA) or male and female primary CD4+ T cells, isolated as above, were cultured in RPMI-1640 supplemented with 1% l -glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 220 μM 2-mercaptoethanol, and 10% FBS (10% RPMI). Generation of MALT1KO Jurkat cells was performed as described40 (link). All mammalian cell cultures were grown at 37 °C and 5% CO2. Escherichia coli strain MAX Efficiency DH5α competent cells (Cat#18258012, Invitrogen, Thermo Fisher Scientific, PA, USA) were used for plasmid transformation and preparation. Transformed E. coli DH5α were grown at 37 °C and 230 rpm shaking.
6
Retroviral Reprogramming of Cells
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Platinum-E (Plat-E) retroviral packaging cells (Cell Biolabs, San Diego, CA, USA) were prepared for plasmid transfections by seeding 8 × 106 per 100mm dish (one dish for each reprogramming gene). Plat-E cells were maintained in Fibroblast-Platinum (FP) medium (Dulbecco’s modified Eagle medium (DMEM), 10% FBS, and 50 U of penicillin-streptomycin). After 24 h, we introduced each pMXs retroviral plasmid DNA (Oct4, Sox2, Klf4, c-Myc, and Ds-Red), (Addgene plasmids 13366, 13367, 13370, 13375 and 22724, respectively) into Plat-E cells using X-tremeGENE 9 DNA transfection reagent (Roche, High River, AB, Canada), according to the manufacturer’s recommendations. Furthermore, 18 μL of X-tremeGENE 9 transfection reagent was added to 300 μL of OptiMEM to a 1.5-mL tube. A total of 8 μg of each retroviral vector was added into the prepared XtremeGENE9-OptiMEM tube drop-by-drop and incubated for 15 min. Each vector–XtremeGENE 9 complex was added dropwise into the Plat-E cell–containing dishes and incubated overnight at 37 °C, 5% CO2. The following day, the medium was replaced with 10 mL of fresh FP medium. Forty-eight hours after transduction, we collected virus-containing medium from each transfection by filtering through a 0.45 μm Acrodisc filter (Pall Life Sciences, Mississauga, ON, Canada).
7
Retroviral Vector Packaging and Osteoclast Differentiation
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Packaging of the retroviral vectors pMX-IRES-EGFP, pMX-cFos-IRES-EGFP and pMX-NFATc1-IRES-EGFP was performed using transient transfection of these pMX vectors (Cell Biolabs, Inc., San Diego, CA, USA) into platinum-E (plat-E) retroviral packaging cells (Cell Biolabs, Inc.) using X-tremeGENE 9 (Roche, Nutley, NJ, USA) according to the manufacturer's protocol. Following incubation at 37°C in fresh medium for 2 days, the culture supernatants of the retrovirus-producing cells were collected. For retroviral infection, non-adherent BMCs were cultured in M-CSF (30 ng/ml) for 2 days. The BMMs were incubated with viral supernatant medium of pMX-IRES-EGFP, pMX-cFos-IRES-EGFP and pMX-NFATc1-IRES-EGFP virus-producing plat-E cells together with polybrene (10 ng/ml) and M-CSF (30 ng/ml) for 6 h. The infection efficiency of the retrovirus was determined by green fluorescent protein (GFP) expression and was always >80%. Post-infection, the BMMs were induced to differentiate in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 days. The expression of each construct was detected using a fluorescence microscope and osteoclast formation was determined by fixing in 3.7% formalin, permeabilizing with 0.1% Triton X-100, and staining with TRAP solution.
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