Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and B cell lymphoma and B-ALL patients after informed consent on protocols approved by the Baylor College of Medicine Institutional Review Board (H-15152, H-27471, H-19384 and H-31970). Capan-1 (pancreatic cancer cell line) and DU145 (prostate cancer cell line) were obtained from the American Type Culture Collection (Rockville, MD). NALM6 (pre-B-ALL cell line) and Raji (Burkitt lymphoma cell line) were gifted by Dr. Maksim Mamonkin. Cells were maintained in a humidified atmosphere containing 5% carbon dioxide (CO2) at 37 °C. Capan-1 and DU145 cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco BRL Life Technologies, Inc., Gaithersburg, MD) while NALM6 and Raji cells were maintained in RPMI-1640 media (GE Healthcare Life Sciences, Pittsburgh, PA). Capan-1 cells were grown in IMDM containing 20% heat-inactivated fetal bovine serum (FBS) (Hyclone, Waltham, MA) with 2 mM L-GlutaMAX (Gibco BRL Life Technologies, Inc.) while other cell lines were grown in their specific media containing 10% FBS with 2 mM L-GlutaMAX.
Rpmi 1640 media
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Austria
RPMI-1640 media is a cell culture medium designed for the in vitro cultivation of a wide variety of cell types. It provides the necessary nutrients and growth factors to support the growth and maintenance of various cell lines. The formulation includes amino acids, vitamins, inorganic salts, and other components to create a balanced and optimized environment for cell growth and proliferation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by GE Healthcare (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Dmem media, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by GE Healthcare (2 mentions)
Non essential amino acids (neaa), by GE Healthcare (2 mentions)
Heat inactivated fcs, by Atlanta Biologicals (2 mentions)
Clone cd28, by BD (2 mentions)
Anti cd3 clone ucht1, by BioLegend (2 mentions)
Negative selection isolation kit, by Miltenyi Biotec (2 mentions)
Anti cd4 clone rpa t4, by BioLegend (2 mentions)
Clone ucht1, by BD (2 mentions)
Mtor inhibitor rapamycin, by Thermo Fisher Scientific (2 mentions)
2 deoxy d glucose 2 dg, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Human il 3, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
L glutamine, by GE Healthcare (2 mentions)
23 protocols using rpmi 1640 media
1
Isolation and Culture of Blood and Cancer Cells
2
Culturing and Implanting Tumor Cell Lines
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B16-F10, TC-1, and B16.OVA were cultured in RPMI-1640 media (GE Life
Sciences), and MC38 was cultured in DMEM media (Gibco), each supplemented with
10% v/v heat-inactivated FCS (Atlanta Biologicals), 100 U/mL penicillin, 100
μg/mL streptomycin (Gibco), 1× non-essential amino acids (GE Life
Sciences), and 1 mM sodium pyruvate (GE Life Sciences). B16.OVA media was
supplemented with 0.5 mg/mL G418. For each tumor cell implantation, a frozen
aliquot was thawed, passaged once, and harvested using trypsin EDTA (Gibco),
quenched with HI-FCS, washed in PBS, and implanted subcutaneously in sterile
PBS. Tumors were measured using digital calipers twice per week, and tumor
volume was estimated using the formula [tumor volume = short × short
× long / 2]. Animals were euthanized when tumors reached size criteria
(1000 mm3 or 2000 mm3).
Sciences), and MC38 was cultured in DMEM media (Gibco), each supplemented with
10% v/v heat-inactivated FCS (Atlanta Biologicals), 100 U/mL penicillin, 100
μg/mL streptomycin (Gibco), 1× non-essential amino acids (GE Life
Sciences), and 1 mM sodium pyruvate (GE Life Sciences). B16.OVA media was
supplemented with 0.5 mg/mL G418. For each tumor cell implantation, a frozen
aliquot was thawed, passaged once, and harvested using trypsin EDTA (Gibco),
quenched with HI-FCS, washed in PBS, and implanted subcutaneously in sterile
PBS. Tumors were measured using digital calipers twice per week, and tumor
volume was estimated using the formula [tumor volume = short × short
× long / 2]. Animals were euthanized when tumors reached size criteria
(1000 mm3 or 2000 mm3).
3
Activation and Metabolic Regulation of Naive T Cells
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Naïve CD4+ T cells were isolated from fresh human blood samples with a negative selection isolation kit from Miltenyi Biotec as per protocol. The purity of naïve CD4+ T cells was evaluated with staining of anti-CD3 (Clone UCHT1, BioLegend, San Diego, USA), anti-CD4 (clone RPA-T4, BioLegend, San Diego, USA), and anti-CD45RA (HI100, BioLegend, San Diego, USA). Isolated cell purities were over 95% (Supplementary Figure S1 ). Cells were cultured in RPMI 1640 media (GE Health Care Life Sciences, Marlborough, USA) supplemented with 10% FBS (Life Technologies, Carlsbad, USA). Cells were stimulated overnight with anti-CD3 (10 μg/mL, pre-coated on plate, Clone UCHT1, BD Biosciences, San Jose, USA) and anti-CD28 (2.5 μg/mL, Clone CD28.2, BD Biosciences, San Jose, USA), with and without glycolysis inhibitor 2-deoxy-d -glucose (2-DG, 2 mg/mL, Acros Organics, New Jersey, USA), mTOR inhibitor rapamycin (100 Nm, Alfa Aesar, Ward Hill, USA) or H2O2 (50 μM, Sigma-Aldrich, St. Louis, USA). The next day the antibodies were removed and fresh media (RPMI and 10% FBS) were added with and without 2-DG, rapamycin, or H2O2. The cells were cultured for a total of 3 days before harvesting.
4
Antifungal MIC Assay for C. auris
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Minimum Inhibition Concentration (MIC) assay was performed on all the 11 C. auris strains following previously published protocol46 (link). Briefly, the C. auris strains were cultured in YPD at 37 °C for overnight, and 0.1 OD of the cultured cells were used as inoculum for MIC the next day. Antifungals fluconazole (FLC), 5- flucytosine (5-FC), amphotericin B (AMB), and micafungin (MF) were two-fold serially diluted in RPMI 1640 media (GE Life Sciences) with 25 mM HEPES (pH 7.0) in a flat bottom 96-well plate (Costar). The plates were incubated for 48 h at 37 °C without shaking. In the plate, one column without any antifungal and one column without cells were used as controls for the assay. MICs were performed in biological triplicate and the average value of the triplicate is reported.
5
Gingival Tissue Isolation for Flow Cytometry
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Gingival tissue was harvested during periodontal surgery from an inflamed site with a persistent periodontal pocket ≥6 mm in periodontitis patients (n = 6). In the controls, gingival tissue was harvested from a site without periodontal pocket in conjunction with an implant surgery or tooth extraction performed for reasons other than periodontitis (n = 6). Tissues were placed in complete RPMI 1640 media (GE Healthcare Life Sciences, Uppsala, Sweden). The tissues were cut into small pieces and incubated with 0.3 mM CaCl2 in phosphate buffered saline (PBS) at 37°C with magnetic stirring for 10 min. After that, they were incubated with Collagenase II (0.5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and DNase I (0.25 mg/ml; Roche, Basel, Switzerland) in RPMI 1640 without FCS at 37°C with magnetic stirring for 40 min. The suspension was filtered through a 70-μm mesh filter (Falcon, Corning, NY, USA), washed with complete RPMI, and centrifuged at 400 g for 5 min. Cell suspensions were then stained and analyzed by flow cytometry.
6
Basophil Activation Assay for IgE Sensitivity
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Basophil activation was performed as previously described33 (link). Buffy coats of human blood from healthy, de-identified, consenting donors were obtained from the MGH Blood Transfusion Service. Peripheral blood mononuclear cells (PBMCs) were separated from buffy coats by a density gradient centrifugation using Ficoll Paque Plus (GE Healthcare) and resuspended in 0.5% BSA in RPMI 1640 Media (GE Healthcare). PBMCs were incubated for 2 min with ice-cold lactic acid buffer (13.4 mM lactate, 140 mM NaCl, 5 mM KCl, pH 3.9) to remove endogenous human IgE on the cell surface prior to neutralization by 12% Tris (pH 8). Cells were then washed and incubated 1 hour at 37°C with 1 μg OVA-specific SiahIgE or AshIgE per 1 × 106 cells in basophil activation buffer (0.5% BSA, 2 mM CaCl2 and 2 mM MgCl2 in RPMI 1640 Media). Sensitized cells were washed and resuspended in basophil activation buffer supplemented with 10 ng/mL human IL3 (PeproTech) prior to 30 min OVA activation. Activation was stopped by addition of ice-cold 0.2 M EDTA in FACS buffer. Cells were washed and resuspended in FACS buffer prior to antibody staining (Extended Data Table 3 ) for activation marker (LAMP-3; CD63+) on basophils (CD123+HLADR−).
7
Establishment and Characterization of Bladder and Canine Transitional Cell Carcinoma Cell Lines
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Human bladder TCC cell lines h-UM-UC-3, h-T24, and h-5637 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines were authenticated via short tandem repeat DNA profiling by Genetica DNA Laboratories (Burlington, NC, USA). h-T24 cells were grown in McCoy’s media (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), h-UM-UC-3 cells were grown in minimum essential media (GE Healthcare Bio-Sciences), and h-5637 cells were grown in RPMI-1640 media (GE Healthcare Bio-Sciences) supplemented with 10% fetal bovine serum, 100 IU penicillin, and 100 μg/mL streptomycin. Canine TCC (K9TCC) cell lines, K9TCC#1Lillie and K9TCC#5Lilly, were established and characterized in the laboratory of Dr Cekanova as described previously in detail.40 (link),48 ,49 (link) K9TCC#1Lillie and K9TCC#5Lilly were grown in complete RPMI-1640 media supplemented with 10% fetal bovine serum, 100 IU penicillin, and 100 μg/mL streptomycin. Cells were grown at 37°C and 5% CO2.
8
Cultivation of Breast Cancer Cell Lines
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Human breast cancer cell lines MDA-MB-453 and HCC-1937 were kindly provided by the Stem Cell Bank, Chinese Academy of Sciences. The cells were cultured in L-15 and RPMI-1640 media (GE Healthcare, Logan, UK), supplemented with 2 mM L-glutamine and 10% FBS (Life Technology, Shanghai, China), containing penicillin (100 U/mL) and streptomycin (100 μg/mL, Life Technology, Shanghai, China). The HCC-1937 cells were cultured in an incubator with 5% CO2 at 37°C and MDA-MB-453 cells were cultured in an incubator at 37°C without CO2 (air, 100%).
9
Culturing Daudi Cell Line
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The Daudi cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA). Daudi cells were maintained in RPMI 1640 media (GE Healthcare Life Sciences, Pittsburgh, PA) supplemented with 10% FBS (GE Healthcare Life Sciences) and 1% GlutaMAX (Thermo Fisher Scientific, Waltham, MA). Cell were grown at 37o C in a humidified atmosphere containing 5% carbon dioxide.
10
Culturing and Implanting Tumor Cell Lines
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