Authenticated THP-1 cells were obtained from the American Type Culture Collection (ATCC, #TIB-202) and maintained in RPMI media supplemented with 10% Fetal bovine serum (Gibco), 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 1 mM HEPES buffer and 1% penicillin and streptomycin. THP-1 cells were originally derived from the peripheral blood of a 1-year-old human male with acute monocytic leukemia. Mouse bone marrow cells were flushed from femurs of male and female mice, differentiated into bone marrow-derived macrophages (BMDM) with L929 cell-conditioned medium (25% v/v; ATCC, #CCL-1), supplemented with 10% heat-inactivated FBS (Gibco) and were analyzed after 6-7 days. L929 cells were originally derived from a male C3H/An mouse. Authenticated human embryonic kidney (HEK) 293 cells (CRL-3216, ATCC) and Lenti-X HEK293 cells (632180, Takara Bio) were maintained in DMEM high glucose media containing 10% FBS, 100 IU mL−1 penicillin and 1 mg mL−1 streptomycin. HEK293 cells were originally derived from a female fetus. Cells were grown at 37°C in 5% CO2 and were routinely tested for mycoplasma contamination (MycoAlert, Lonza).
Lenti x hek293 cells
Manufactured by Takara Bio
Lenti-X HEK293 cells are a stable, highly transfectable human embryonic kidney cell line specifically engineered for the production of lentiviral vectors. These cells provide a reliable and efficient system for the generation of high-titer lentiviral particles.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Tib 202, by Thermo Fisher Scientific (2 mentions)
Ccl 1, by American Type Culture Collection (2 mentions)
Crl 3216, by American Type Culture Collection (2 mentions)
Mycoalert, by Lonza (2 mentions)
Macs cd34 microbead kit ultra pure, by Miltenyi Biotec (2 mentions)
Hyclone, by Thermo Fisher Scientific (2 mentions)
32d cells, by American Type Culture Collection (2 mentions)
Interleukin 3 (il 3), by American Type Culture Collection (2 mentions)
Polybrene, by Merck Group (1 mentions)
Fugene, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
6 protocols using lenti x hek293 cells
1
Cell Culture Protocols for Immunological Research
2
Lenti-X HEK293 Cell Transduction
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Briefly, Lenti-X™ HEK293 cells (Takara Bio) were transfected with pMDLg/pRRE, pMD2.G, pRSV-Rev plasmids (all kind gifts from Angeliki Malliri, Cancer Research UK Manchester Institute) and pCDH-EF1α-DGAT1-T2A-mApple viral vectors using Fugene (Promega) following standard protocols. The viral containing supernatant was filtered using a 0.45 μm filter and frozen at −80°C prior to transduction of target cells. The supernatant containing the viral particles was added to target cells along with 10 ng/mL Polybrene (Millipore) for 24 h. Target cells were then grown and selected from single cell colonies.
3
Cell Culture Protocols for Immunological Research
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Authenticated THP-1 cells were obtained from the American Type Culture Collection (ATCC, #TIB-202) and maintained in RPMI media supplemented with 10% Fetal bovine serum (Gibco), 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 1 mM HEPES buffer and 1% penicillin and streptomycin. THP-1 cells were originally derived from the peripheral blood of a 1-year-old human male with acute monocytic leukemia. Mouse bone marrow cells were flushed from femurs of male and female mice, differentiated into bone marrow-derived macrophages (BMDM) with L929 cell-conditioned medium (25% v/v; ATCC, #CCL-1), supplemented with 10% heat-inactivated FBS (Gibco) and were analyzed after 6-7 days. L929 cells were originally derived from a male C3H/An mouse. Authenticated human embryonic kidney (HEK) 293 cells (CRL-3216, ATCC) and Lenti-X HEK293 cells (632180, Takara Bio) were maintained in DMEM high glucose media containing 10% FBS, 100 IU mL−1 penicillin and 1 mg mL−1 streptomycin. HEK293 cells were originally derived from a female fetus. Cells were grown at 37°C in 5% CO2 and were routinely tested for mycoplasma contamination (MycoAlert, Lonza).
4
Lentiviral-Mediated VAV3 Overexpression
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pRRL-HA-VAV3WT was constructed by cloning VAV3 from pC.HA-VAV3 (14555, Addgene) into the lentiviral vector pRRL-IRES-RFP (University of California, Los Angeles, Vector Core) by using Gibson Assembly. For the Vav3 DH domain mutated form, pLVX-Myc-VAV3N369A was used. The sequence of Rap1S17N was provided by X. Zhang (Southwestern University, Dallas, TX) as part of pcDNA3-HA-Rap1-S17N. Lentiviral particles were generated by transfecting Lenti-X HEK293 cells (Takara Bio Inc.) with target, VSV-G pseudotype, and delta8.2 packaging plasmids by using Lipofectamine 2000 (Invitrogen). Conditioned medium was collected after 48 h and passed through 0.45-µm filters. ECs were transduced at 50% confluency overnight in EGM-2 medium containing protamine 4 µg/ml sulfate. Empty pRRL-IRES-RFP was used as control vector.
5
Culturing and Isolation of Hematopoietic Cells
6
Culturing and Isolation of Hematopoietic Cells
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OP9 cells (a gift from the Mikkola Laboratory, UCLA) were cultured in alpha minimum essential medium (aMEM) with 2 mM L-glutamine, 1% pen-strep, 20% Hyclone (Thermo Fisher Scientific) fetal bovine serum (FBS). For OP9/leukocyte co-cultures, media was supplemented with 5 ng/mL thrombopoietin (TPO), 50 ng/mL SCF, 10 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3L), 5 ng/mL interleukin-6 (IL-6), and 5 ng/mL IL-3 (Peprotech). 32D cells (CRL-11346 American Type Culture Collection [ATCC]) were cultured according to ATCC recommendations with 10 ng/mL IL-3. Lenti-X HEK293 cells (632180 Clonetech) were cultured in DMEM 10% FBS. G1E-ER4 cells (a gift from the Ganz Laboratory, UCLA) were cultured with tamoxifen according to Rylski et al. (2003) (link). BV173, KCL22, and K562 cells (a gift from the Colicelli Laboratory, UCLA), Tf1a (ATCC CRL-2451) and HEL 92.1.7 (ATCC TIB-180) were all cultured according to ATCC recommendations.
Human PBMC and CD34+ cord blood cells were purchased from the UCLA/Core Facility Research (CFAR) Virology Core Laboratory. Mononuclear cells were isolated using a Ficoll gradient. Monocytes (PBMC fraction adhering to a TC plate) and lymphocytes (non-adhered portion) were used for RNA. For cord blood, CD34+ (positive selected) and CD34− (negative selected) blood cells were isolated using human Miltenyi MACS CD34 MicroBead Kit Ultra Pure and used for RNA.
Human PBMC and CD34+ cord blood cells were purchased from the UCLA/Core Facility Research (CFAR) Virology Core Laboratory. Mononuclear cells were isolated using a Ficoll gradient. Monocytes (PBMC fraction adhering to a TC plate) and lymphocytes (non-adhered portion) were used for RNA. For cord blood, CD34+ (positive selected) and CD34− (negative selected) blood cells were isolated using human Miltenyi MACS CD34 MicroBead Kit Ultra Pure and used for RNA.
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