K562 and C1R cell lines were obtained from ATCC (Manassas, VA, USA). The genetically modified clone of K562 cells expressing membrane-bound IL-21 (К562-mbIL21) was kindly provided by Dr Dean Lee (MD Anderson Cancer Center, USA). This clone is characterized as tCD19+CD64+CD86+CD137L+. All cell lines were cultivated in the following culture medium: RPMI-1640 (PanEco, Russia) supported with 10% FCS (fetal calf serum) (HyClone, USA), 2 mmol L−1 of l -glutamine (PanEco) and Antibiotic Antimycotic Solution (Sigma-Aldrich, St. Louis, MO, USA). Surface expression of IL-21 was tested periodically by flow cytometry. K562-mbIL21 cells and unmodified K562 cells were irradiated with γ radiation (100 Gy), cryopreserved at −150°C and then recovered prior to NK cell stimulation.
Rpmi 1640
Manufactured by PanEco
Sourced in United States
RPMI-1640 is a cell culture medium commonly used for the in vitro cultivation of various cell types, including human and animal cells. It provides the necessary nutrients and growth factors to support cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by PanEco (6 mentions)
Dulbecco modified eagle medium (dmem), by PanEco (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Trypsin edta solution, by PanEco (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Antibiotic antimycotic solution, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by PanEco (2 mentions)
Penicillin, by PanEco (2 mentions)
Streptomycin, by PanEco (2 mentions)
Recombinant human il 2, by Roche (2 mentions)
Pen strep glut, by PanEco (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by PanEco (2 mentions)
Ficoll, by PanEco (1 mentions)
Fura red am, by Thermo Fisher Scientific (1 mentions)
Cd62p alexa647, by Sony (1 mentions)
Cd11b fitc, by Sony (1 mentions)
Cd66b pe, by Sony (1 mentions)
Mrs2179, by Bio-Techne (1 mentions)
Phosphate buffered saline tablets, by PanEco (1 mentions)
27 protocols using rpmi 1640
1
K562 and C1R Cell Lines for NK Cell Stimulation
2
Platelet Activation and Signaling Pathways
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The sources of the materials were as follows: calcium-sensitive cell-permeable fluorescent dye Fura-Red-AM, (Molecular Probes, Eugene, OR); LPS O111:B4, LPS O127:B8, ADP, PGI2, EGTA, HEPES, bovine serum albumin, apyrase grade VII, TRAP-6 (SFLLRN) (Sigma-Aldrich, St Louis, MO); CD62p-Alexa647, CD11b-FITC, CD66b-PE (Sony Biotechnology, San Jose, USA); HBSS, RPMI 1640, Ficoll (PanEco, Moscow, RF), MRS2179, Mes-AMP (Tocris Bioscience, UK); Cysteine-containing version of cross-linked collagen-related peptide (CRP) was kindly provided by Prof. R.W. Farndale (the University of Cambridge, Cambridge, UK). Tyrode’s buffer (150 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.4 mM NaH2PO4, 0.4 mM Na2CO3, 5 mM HEPES, 5 mM glucose, 0.5% BSA, pH 7.35) was fresh made from reagents (Sigma-Aldrich, St Louis, MO). IBMX was a kind gift of prof. S.P. Gambaryan (SPBU, St. Petersburg, Russia).
3
Antimicrobial Peptide MIC Assay
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Minimum inhibitory concentrations (MICs) were detected in a serial dilution assay with the purified compounds according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocol [49 (link)]. Tests were carried out by taking a 100 mL stock solution of the peptide in a two-fold serial dilution at concentrations ranging from 0.5 to 64 mg/mL (0.1–12 µM) in DMSO (Merck, Darmstadt, Germany). The assays were conducted in 96-well microtiter plates (BioCell Technology, Irvine, CA, USA) in RPMI 1640 (PanEco, Moscow, Russia) medium without the addition of Na2CO3. Amphotericin B (AmB) was used as a positive control; the nutrient medium was used as a negative control. Each experiment was carried out in triplicate. MIC values were defined as the lowest concentration of compounds at which the microorganisms treated demonstrated no visible growth after 48 h of incubation. Assays were performed three times in triplicate
4
Amniotic Fluid Karyotyping Protocol
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For karyotype analysis, 10–20 ml of amniotic fluid was obtained by amniocentesis. The amniotic fluid cells with 4.5 ml medium (RPMI-1640, Paneco, Russia) were cultured in a 37 °C incubator with 5% carbon dioxide. The cells were harvested at 10–12 days. After colchicine treatment for 2 h, the cells were digested using 1:250 trypsin, and incubated with 0.075 M KCl for 30 min. The prefixation, fixation, dropping, baking, and G-band staining were performed next. A total of 100 dividing phases were counted using an all-chromosome image analysis system based on the “An International System for Human Cytogenetic Nomenclature, ISCN2016”.
5
Cytokine Activation in Spleen Cells
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In order to activate cytokines synthesis and secretion we cultivated 106/mL spleen cells in 1 mL of culture medium with concanavalin A (5 mkg/mL) for 20 hours at 37°C and 5% CO2 in 24-well cultured plates. The culture medium consists of RPMI-1640 (PanEco, Russia), 5% of inactivated fetal calf serum, 2 mM of glutamine, and 50 mkg/mL of gentamicin.
6
Evaluating PBMC Viability in Culture
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The viability of human peripheral blood mononuclear cells was studied.
At first, 10 mL of blood was diluted 1:1 with PBS, layered on ficoll and centrifuged at 2000 rpm for 30 min. Thus, a layer containing mononuclear cells was obtained. Then, 5 mL of the buffy coat sample were washed twice with 10 mL of PBS, centrifuged at 1500 rpm for 5 min, and placed in a 12-well plate containing 2 × 106 cells/mL of RPMI-1640 (Paneco, Moscow, Russia). We added to this 10% fetal bovine serum (Paneco, Moscow, Russia), antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin) and 2 mM L-glutamine at 37 °C in a 5% CO2 atmosphere. The studied samples were placed in a 12-well plate and incubated for 24 and 72 h. The viability of this product was then determined using the trypan blue dye exclusion method.
At first, 10 mL of blood was diluted 1:1 with PBS, layered on ficoll and centrifuged at 2000 rpm for 30 min. Thus, a layer containing mononuclear cells was obtained. Then, 5 mL of the buffy coat sample were washed twice with 10 mL of PBS, centrifuged at 1500 rpm for 5 min, and placed in a 12-well plate containing 2 × 106 cells/mL of RPMI-1640 (Paneco, Moscow, Russia). We added to this 10% fetal bovine serum (Paneco, Moscow, Russia), antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin) and 2 mM L-glutamine at 37 °C in a 5% CO2 atmosphere. The studied samples were placed in a 12-well plate and incubated for 24 and 72 h. The viability of this product was then determined using the trypan blue dye exclusion method.
7
Isolation and Activation of Splenic Cells
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For isolation of splenic cells, a piece of spleen was aseptically removed from each rat, placed in Potter homogenizer containing the Roswell Park Memorial Institute (RPMI)-1640 medium, and single-cell suspensions were prepared. The red blood cells were lysed with distilled water. To activate cytokine synthesis and secretion, we cultivated 106/mL spleen cells in 1 mL of culture medium with concanavalin A (5 µg/mL) for 20 hours at 37°C and 5% CO2 in 24-well cultured plates. The culture medium consisted of RPMI-1640 (PanEco, Moscow, Russia), 5% inactivated FBS, 2 mM glutamine, and 50 µg/mL gentamicin.42 The cell viability was determined according to trypan blue exclusion.
8
Cell Culture Conditions and Maintenance
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Cell lines were obtained from Blokhin CRC cell collection. Cells were cultured in DMEM (for adhesion cell cultures) or RPMI-1640 (for suspension cell cultures) supplemented with L-glutamine (0.584 mg/mL), penicillin (50 U/mL) and streptomycin (50 μg/mL) (PanEco, Moscow, Russia) and 10% fetal bovine serum (PanEco, Russia). Cell lines were incubated at 37 °C in 5% CO2.
9
Isolation and Activation of Rat Splenic Cells
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Isolation and cultivation of splenic cells were carried out as describe in10 (link). For isolation of splenic cells, a piece of spleen was aseptically remove from each rat, placed in Potter homogenizer containing the Roswell Park Memorial Institute (RPMI) 1,640 medium and single-cell suspensions were prepared. The red blood cells were lysed by distilled water. To activate cytokine synthesis and secretion, we cultivated 106/ml spleen cells in 1 ml of culture medium with concanavalin A (5 µg/ml) for 20 h at 37 °C and 5% CO2 in 24-well cultured plates. The culture medium consisted of RPMI-1640 (PanEco, Russia), 5% inactivated foetal bovine serum (FBS), 2 mM glutamine and 50 µg /ml gentamicin75 . The cell viability was determined according to trypan blue exclusion6 (link).
10
Colorectal Cancer and Leukemia Cell Lines
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Human cancer cell lines of colorectal carcinoma HCT116 and Caco-2 were from ATCC (Washington, USA); cell lines of colorectal carcinoma HT-29 and T-lymphoblastic leukemia Jurkat were from the Institute of Cytology, Russian Academy of Sciences (St. Petersburg, Russia). Cell culture media (DMEM, RPMI1640), 0.05% trypsin-EDTA solution, and phosphate-buffered saline tablets were from PanEco (Russia). Fetal bovine serum was from HyClone (USA). WST1 was from Sigma-Aldrich (USA).
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