On day 1, cells were seeded into NunclonTM Delta Surface plates (Nunc, Roskilde, Denmark), and transfected twice, on days 2 and 3, serum-free with either 25 or 50 nM of prevalidated siRNAs specific for RAC1B [6 (link)], SMAD2, SMAD3 (both from Dharmacon, Lafayette, CO, USA), BGN [7 (link)], or the respective scrambled controls, and/or pcDNA3-based expression vectors for SMAD3, SMAD3-mut3A, or SMAD2, for 4 h using Lipofectamine 2000 (Life Technologies). Additional validation of these siRNAs was performed for RAC1B [6 (link)] and SMAD2/SMAD3 (this study).
Nunclon delta surface plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States
Nunclon Delta Surface plates are cell culture plates designed for optimal cell attachment and growth. They feature a proprietary surface treatment that enhances cell adhesion and proliferation. The plates are available in a variety of sizes and well configurations to meet different experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Smad3, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Raw 264.7 murine macrophage, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Incucyte live cell analysis system, by Sartorius (1 mentions)
Doxycycline, by Takara Bio (1 mentions)
Cell culture dishes, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Mithras lb940 plate reader, by Berthold Technologies (1 mentions)
Quanti luc, by InvivoGen (1 mentions)
Multiscreen solvinert filter plate, by Merck Group (1 mentions)
Il 15, by Immunotools (1 mentions)
Cell proliferation dye efluor 670, by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Immunotools (1 mentions)
18 protocols using nunclon delta surface plate
1
Genetic Manipulation of Cellular Pathways
2
Transfection-based RAC1B knockdown
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On day 1, cells were seeded into NunclonTM Delta Surface plates (Nunc, Roskilde, Denmark), and transfected twice, on days 2 and 3, serum-free with either 50 nM of prevalidated siRNAs specific for RAC1B [18 (link)] or a scrambled control for 4 h using Lipofectamine 2000 (Panc1 cells) or Lipofectamine RNAiMAX (Capan1 and Colo357 cells) (both from Life Technologies). The procedure for plasmid vectors encoding HA-RAC1B (in β) or MYC-RAC1-L61 (in pRK5) was identical except that cells were transfected only once Afterwards, cells received normal growth medium and were incubated for another 24 or 48 h prior to qPCR, immunoblot analysis, or real-time migration assay.
3
Transient Transfection of Signaling Regulators
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For transient transfection, cells were seeded on day 1, cells into NunclonTM Delta Surface plates (Nunc, Roskilde, Denmark), and transfected twice, on days 2 and 3, with either 25 or 50 nM of prevalidated siRNAs specific for RAC1B [24 (link)], SMAD3 (both from Dharmacon, Lafayette, CO, USA), or the respective scrambled controls. The TGFB1 siRNA was provided by Qiagen (cat.# 1027416, a mixture of four different pre-evaluated siRNAs), and the SMAD3 siRNA by Dharmacon (Lafayette, CO, USA). Additional validation of these siRNAs was performed for RAC1B [24 (link)] and SMAD3 [18 (link)]. An expression vector for full length human TGFβ1 (cat.# SC119746) was purchased from OriGene Technologies Inc. (Rockville, MD, USA), while a MYC-tagged version of constitutively active RAC1 (Q61L mutation) in the pRK5 vector was a kind gift of G.M. Bokoch (La Jolla, CA, USA). SiRNAs or plasmids were mixed with Lipofectamine 2000 (Life Technologies) according to the manusfacturers recommendations and transfected in cells serum-free for 4 h. Following transfection with siRNAs and another 24-h incubation in normal growth medium, cells were trypsinized, diluted in trypan blue solution and viable (non-blueish) cells counted with a Neubauer chamber.
4
Transient Transfection of siRNA and Plasmids
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For transient transfection of siRNA and DNA plasmids, cells were seeded on day 1 in NunclonTM Delta Surface plates (Nunc, Roskilde, Denmark) and transfected twice, on days 2 and 3, serum-free with 25 or 50 nM of siRNA specific for RAC1B, PAR2, or ALK5 [8 (link),9 (link)], or the respective scrambled control siRNAs, for 4 h using Lipofectamine 2000 (Life Technologies). Additional validation of these siRNAs was performed for RAC1B [2 (link)], and ALK5 and PAR2 [8 (link),9 (link)]. A PAR2 expression vector (PAR2-Myc-DKK) was purchased from Origene (Rockville, MD, USA).
5
Investigating p73 and SMAD4 in Pancreatic Cancer
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On d 1, PANC-1 or HPAFII cells were seeded into NunclonTM Delta Surface plates (Nunc, Roskilde, Denmark) and transfected twice, on d 2 and 3, serum-free with 50 nM of pre-validated small interfering RNAs (siRNAs) specific for p73 or SMAD4, or scrambled siRNAs as control for 4 h, using Lipofectamine 2000 (PANC-1) or RNAiMAX (HPAFII) (both from Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer‘s instructions. Transfected cells were subjected to immunoblot analyses, reporter gene assays, or cell migration assays. For reporter gene assays, PANC-1 cells were seeded in 96-well plates on d 1 and co-transfected on d 2 serum-free with various siRNAs and Lipofectamine 2000. Twenty-four h later (d 3), cells again received Lipofectamine 2000 with the same siRNAs together with p(CAGA)12 MLP-Luc and the Renilla luciferase encoding vector pRL-TK-Luc. On d 4, cells were treated with TGF-β1 for 24 h and luciferase activities were determined with the Dual Luciferase Assay System (Promega, Mannheim, Germany). In all reporter gene assays, the data were derived from 6 wells processed in parallel and normalized with Renilla luciferase activity as described in detail elsewhere [7 (link)].
6
Culturing Murine Macrophages in Supplemented DMEM
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RAW264.7 murine macrophages (American Type Culture Collection, ATCC) were seeded in 12 or 24-well Nunclon Delta Surface plates (Thermo Fisher Scientific, MA, United States) containing Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) plus 100 UI penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B (antibiotic-antimycotic solution, Thermo Fisher Scientific). Cells were incubated at 37°C in a 5% CO2 environment for the necessary time in accordance with the experiments described below.
7
Cultivation and Isolation of Leukemia Cell Lines
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HL-60 human promyelocytic- and THP-1 human monocytic leukaemia cell lines were acquired from European Cell Culture Collection. Cells were cultivated in RPMI-1640 media supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1% 2-[4-(2-hydroxyethyl)piperazin-1-yl]etansulfonic acid HEPES buffer (all reagents from Sigma Aldrich, St. Louis, MO, USA). Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood, obtained by venepuncture from a 43-year-old female donor, following her informed written consent. PBMCs were isolated in density gradient, using Histopaque 1077 separation media and Hanks Modified Eagle’s Media (both from Sigma Aldrich) for cell wash, using our previously described method [36 (link)].
For colorimetric testing, cells were seeded on 96-well micro plates, at a concentration of 3 × 104 cells/100 μL media, and incubated for 24 h (Nunclon Delta surface plates from Thermo Fischer Scientific, Waltham, MA, USA). For fluorescence measurements, black, clear-bottom 96-well plates were used (from Corning, Tewksbury, MA, USA).
For colorimetric testing, cells were seeded on 96-well micro plates, at a concentration of 3 × 104 cells/100 μL media, and incubated for 24 h (Nunclon Delta surface plates from Thermo Fischer Scientific, Waltham, MA, USA). For fluorescence measurements, black, clear-bottom 96-well plates were used (from Corning, Tewksbury, MA, USA).
8
Cell Viability Assay with Radiation and Drugs
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Cells were plated at a density of 3000 cells/well in 96-well Nunclon Delta Surface plates (Thermo Fisher Scientific). Following overnight incubation, cells were treated with either DCA, erlotinib, rociletinib or DCA in combination with erlotinib or rociletinib. After radiation and/or after treatment the 96well plates were placed in a Incucyte® Live Cell Analysis system (Incucyte ZOOM 2016B, EssenBioscience Ltd., UK) and percent confluency was estimated using the Incucyte Zoom software and normalized to the first time point of each cell type or DMSO control as described in figure legends. The resazurin cell viability assay was used to measure cell viability by using fluorescence to detect cellular metabolic activity. After indicated treatment period (72 h), resazurin was diluted in medium (10% v/v) and added to the cells. The cells were further incubated for 3 h before fluorescence was measured using a Tecan SPARK instrument (Tecan Trading AG, Switzerland).
9
Isolation and Culture of iKPC Cells and CAFs
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All cells were cultured in a humidified incubator at 37°C with 5% CO2. GFP-positive iKPC cells were flow-sorted and cultured in DMEM + 10% Tet-approved FBS (Clontech [which now belongs to Takara] 631368) + penicillin–streptomycin (Gibco 15140122) with 1 μg/mL doxycycline (Takara 631311) in cell culture dishes (Falcon). For Matrigel-based 3D cell culture, 2000–5000 iKPC cells were suspended in Matrigel and plated in 24-well Nunclon Delta Surface plates (Thermo) using the same culture media as 2D culturing. Flow-sorted primary CAFs (GFP−, CD45−, CD31−, and PDPN+) were cultured in the same condition described above but without doxycycline, and the cells were cultured for less than three passages before conditioned media collection, coinjection, or Western blot analysis. To establish spontaneously immortalized CAF1, pCAF1 cells were cultured in cell culture dishes with the media changed every week until the cells overcame the initial growth stall in ∼4 wk.
10
TEM-1 β-Lactamase Activity Assay
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TEM-1 β-lactamase activity was assayed as described previously [6 (link)]. Briefly the rate of
nitrocefin (50 μM) hydrolysis was observed at 486 nm at 25°C for 60 minutes
in 10 mM HEPES, 300 mM NaCl, 10% glycerol (pH 7.0) using 10 nM native
protein, native protein in 0.5 M GdnCl and refolded protein in 0.5 M GdnCl.
Activity measurements were carried out on Varioskan Flash (ThermoScientific)
using Nunclon delta surface plates (ThermoScientific).
nitrocefin (50 μM) hydrolysis was observed at 486 nm at 25°C for 60 minutes
in 10 mM HEPES, 300 mM NaCl, 10% glycerol (pH 7.0) using 10 nM native
protein, native protein in 0.5 M GdnCl and refolded protein in 0.5 M GdnCl.
Activity measurements were carried out on Varioskan Flash (ThermoScientific)
using Nunclon delta surface plates (ThermoScientific).
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