Human THP-1 monocyte cell line (ATCC® TIB-202TM) was obtained from ATCC (Manassas, VA, USA). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, Thermo Fisher Scientific Inc., Waltham, MA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (GIBCO, Thermo Fisher Scientific Inc., Waltham, MA, USA). Cells were cultured in 5% CO2-humidified atmosphere at 37 °C and passaged twice a week. THP-1 cells were differentiated by addition of phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO, USA) to the culture medium at a final concentration of 100 nM for 24 h. Human umbilical vein endothelial cells (HUVECs) were purchased from LONZA Biologics (LONZA Biologics, Cambridge, UK), and maintained in Endothelial Basal Medium-2 supplemented with 2% FBS and growth factors. Cells were incubated in a humidified, 37 °C incubator under 5% CO2. Cell passages between 3 and 5 were used in the present study.
Atcc tib 202tm
Manufactured by American Type Culture Collection
Sourced in United States
The ATCC® TIB-202TM is a cell line originally derived from the spleen of a C57BL/6 mouse. It is commonly used as a source of murine B lymphocytes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Nlrp3, by InvivoGen (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by American Type Culture Collection (1 mentions)
Crl 3304, by American Type Culture Collection (1 mentions)
4 protocols using atcc tib 202tm
1
Cultivation of THP-1 and HUVEC Cells
2
THP-1 Cell Line Culture and Differentiation
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The human acute monocytic leukemia cell line THP-1 (ATCC® TIB-202TM) from the American Type Culture Collection (ATCC®, Manassas, VA, USA) was cultured in RPMI 1640 containing 10% fetal bovine serum (FBS) with a supplement of penicillin–streptomycin (Sigma Aldrich; St. Louis, MO, USA). Wild type (wt) THP-1 cells and the two THP-1 knockout cell lines NLRP3− or ASC− (InvivoGen, Toulouse, France) were used in some experiments. Thus, the gene deletion cell lines lacked key inflammasome components. The cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C. In the regular assays, the THP-1 cells were stimulated 24 h in the presence of 50 nM phorbol 12-myristate 13-acetate (PMA) followed by an additional 24 h period before the cells were exposed to different test stimuli. PMA stimulates differentiation of the non-adherent native monocyte-like THP-1 cell line into an adherent macrophage-like phenotype. In experimental set-ups that involved flow cytometric characterizations, the non-adherent native phenotype of the THP-1 cells was used.
3
THP-1 Cell Culture Protocol
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THP-1 cells (ATCC® TIB-202TM) were purchased from the American Type Culture Collection and cultured according to their specific indications, using an RPMI 1640 medium supplemented with non-heat-treated 10% fetal bovine serum (FBS; Invitrogen), 2 mM L -glutamine, 0.05 mM β-mercaptoethanol, 10 mM HEPES, 4500 mg/L glucose, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. THP-1 cells were kept at a minimum density of 3 × 105 cells/ml and were passaged when reaching 8 × 105 cells/ml. Upon thawing, cells were initially expanded by adding a volume of fresh medium every 48 h until they reached the above-mentioned maximum density, after which they were passaged every 2 days with a complete medium replacement.
4
Differentiation and Treatment of Immune Cell Lines
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The immortalized human monocyte cell line THP-1 (ATCC, #:ATCC® TIB-202TM, obtained from the Tissue culture facility at UNC Chapel Hill), was cultured in RPMI 1640 with L-Glutamine-Bicarbonate (Sigma-Aldrich # R8758) containing 10% FBS (Thermo Fisher Scientific, GibcoTM # 10500064), and 1% penicillin/streptomycin (Sigma-Aldrich #P4333) in an atmosphere of 5% CO2 at 37 °C. To differentiate THP-1 monocytes into macrophages, cells were plated at a density of 1.0 × 106 cells/well in 6-well plates and incubated with 25 μM PMA (12-O-tetradecanoylphorbol) for 24 h. Macrophages were then allowed to rest for 72 h before Aβ or LPS/INFγ treatment as described below. The HMC3 immortalized microglia cell line (obtained from ATCC # CRL-3304) and the astrocyte-like U-87 cell line (ATCC # 30-2003 obtained from the Tissue culture facility at UNC-Chapel Hill) were both cultured in EMEM (ATCC # 30-2003) containing 10% FBS and 1% penicillin/streptomycin in an atmosphere of 5% CO2 at 37 °C. Cells were treated with 1 μM Aβ or 10 ng/ml LPS/20 ng/ml INFγ (Sigma-Aldrich # L2630 /Peprotech # 300-02) during 24 h. Treatments were carried on in serum-free media with 1% penicillin/streptomycin (100 U/ml penicillin/100 μg/ml streptomycin) unless otherwise stated.
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