Assembling of cells and particles to microtissues followed a previously described protocol [34 (link)]. Briefly, hMSC (Lonza, Basel, Switzerland) were cultivated in DMEM low glucose (Sigma Aldrich, Seelze, Germany), 10% (v/v) fetal bovine serum albumin (FBS, Biochrome, Berlin, Germany), 1% amino acids and antibiotics and detached the cells with Trypsin/EDTA (Sigma Aldrich, Seelze, Germany). We added 100 µL of cell suspension (105 cells ml−1) to 100 µL of cGM suspension (0.64 mg ml−1) in Opti-MEM™ with 10% FBS per well of a low adhesion 96-well plate (Spheroid microplate, Corning, NY, USA). After 24 h, the medium was changed to osteogenic medium (OM) based on DMEM, 10% (v/v) FBS, 100 ng/mL Dexamethasone (Sigma Aldrich, Seelze, Germany), 50 µg/mL ascorbic acid (Sigma Aldrich, Seelze, Germany), 10 mM β-Glycerophosphate disodium salt hydrate (Sigma Aldrich, Seelze, Germany) with (OM+BMP-2)/without (OM) 100 ng/mL bone morphogenetic protein (BMP-2, R&D Systems, Wiesbaden, Germany). Medium was changed partially by exchanging 200 µL of culture medium twice for three times a week in order to keep cGM/hMSC and microtissues in the well.
Opti mem
Manufactured by Corning
Sourced in United States, China
Opti-MEM is a cell culture medium developed for the maintenance and growth of a variety of cell lines. It is formulated to provide a nutrient-rich environment that supports cell viability and proliferation. Opti-MEM is designed to be used in a variety of cell culture applications, including transfection experiments and other cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Pspax2, by Addgene (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Pcmv vsv g, by Addgene (5 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Matrigel, by Corning (3 mentions)
Opti mem, by Addgene (3 mentions)
Spheroid microplate, by Corning (2 mentions)
Plv ef1α mcs ires bsd vector, by Biosettia (2 mentions)
Transit lti transfection reagent, by Mirus Bio (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
β glycerophosphate disodium salt hydrate, by Merck Group (1 mentions)
Dmem low glucose, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Bmp 2, by R&D Systems (1 mentions)
Lipofectamine 2000, by Corning (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
29 protocols using opti mem
1
Engineered Bone Microtissue Formation
2
PARG Knockdown in Cells
3
Lentiviral Transduction Protocol for 293T Cells
4
Knockdown of Egr1 in Pluripotent Stem Cells
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Cells were transfected with siRNAs of Egr1 (Thermo Fisher Scientific) at a final
concentration of 10 nM using Lipofectamine® RNAiMAX Transfection Reagent (13778030; Thermo
Fisher Scientific) in accordance with the manufacturer’s protocol. For a 6-well plate (9.6
cm2), we used 4 µL of transfection reagent in 150 µL of OptiMEM (Thermo
Fisher Scientific), 0.8 µL of 50 µM siRNA solution in 150 µL of OptiMEM, and 2 ×
105 PSCs in 2 mL of Essential 8 medium on Matrigel-coated dishes
(hESC-qualified, 354277; Corning, NY, USA). Silencer® Select Negative Control No. 1
(Thermo Fisher Scientific) was used as a control. The medium was changed after 48-h
incubation.
concentration of 10 nM using Lipofectamine® RNAiMAX Transfection Reagent (13778030; Thermo
Fisher Scientific) in accordance with the manufacturer’s protocol. For a 6-well plate (9.6
cm2), we used 4 µL of transfection reagent in 150 µL of OptiMEM (Thermo
Fisher Scientific), 0.8 µL of 50 µM siRNA solution in 150 µL of OptiMEM, and 2 ×
105 PSCs in 2 mL of Essential 8 medium on Matrigel-coated dishes
(hESC-qualified, 354277; Corning, NY, USA). Silencer® Select Negative Control No. 1
(Thermo Fisher Scientific) was used as a control. The medium was changed after 48-h
incubation.
5
Large-Scale Lentiviral Vector Production
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Pooled library virus was made using the same large scale T175 flask method used previously [19 (link)]. Briefly, 24 hours pre-transfection, 18 × 106 HEK293T cells were seeded into a 175 cm2 tissue culture flask with 24 mL of DMEM + 10% FBS. Next day, one solution of Opti-MEM (Corning, 6 mL) and LT1 (Mirus, 305 μL) was combined with a DNA mixture of the packaging plasmid pCMV-VSVG (Addgene 8454, 5 μg), psPAX2 (Addgene 12260, 50 μg), and sgRNA-containing vector (pPapi, 40 μg). This mixture was incubated for 20–30 min at room temperature, during which media was changed on the HEK293Ts. Following incubation, the transfection mixture was added dropwise to cells. The cells were incubated for 6–8 h, after which time media was replaced with DMEM + 10% FBS, supplemented with 1% BSA. 36 hours post-media replacement, virus was harvested.
6
Overexpression of miR-106b-3p in HL-1 cells
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MiR-106b-3p mimics and mimics NC were synthesized by Sangon, China. HL-1 cells seed on six-well plates at a density of 2 × 105 cells/well. When reaching about 80–90% confluence, cells were transfected with miR-106b-3p mimics or mimics NC using Lipofectamine™ 2000 (Invitrogen, Waltham, MA). MiR-106b-3p mimics, mimics NC, and Lipofectamine™ 2000 regent were diluted in 45 µL OPTI-MEM (CORNING). After that, diluted miR-106b-3p mimics was mixed with diluted Lipofectamine™ 2000 regent for 20 min, and then added into HL-1 cells. Cultured for another 24 h and transfection efficiency was measured using RT-qPCR.
7
Lentiviral Vector Production and Knockdown
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Next, 0.75 µg of the target plasmid pLVX-CMV-lncRNA-EGFP-IRES-Puro (Addgene, China), 0.75 µg of psPAX2 (Addgene), and 0.5 µg of pMD2.G (Addgene) were mixed and co-transfected into 293FT cells (which were used to package lentiviruses) using the Effectene Transfection Reagent (QIAGEN, Germany; 301,425). The supernatant of 293FT cells was collected and filtered to harvest the lentivirus. Next, 30 µL of polybrene (10 mg/mL) and 150 µL of lentivirus were added to a 6-well plate containing SW480 cells. Finally, stable cell lines were obtained by selection with puromycin and examined using cell immunofluorescence.
The knockdown of inhibin subunit beta C (INHBC) expression was performed using a siRNA purchased from GenePharma (China). INHBC siRNA or control siRNA (5 µL of siRNA was diluted in 45 µL of OPTI-MEM (CORNING, China)) was transfected into SW480 cells using Lipofectamine™ 2000 (Invitrogen). The sequences of the siRNAs are listed in Additional file3 : Table S1.
The knockdown of inhibin subunit beta C (INHBC) expression was performed using a siRNA purchased from GenePharma (China). INHBC siRNA or control siRNA (5 µL of siRNA was diluted in 45 µL of OPTI-MEM (CORNING, China)) was transfected into SW480 cells using Lipofectamine™ 2000 (Invitrogen). The sequences of the siRNAs are listed in Additional file
8
Lentiviral and Retroviral Production
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HEK293T cells were transfected at 60% confluence using Fugene HD (Promega) in OptiMEM (Corning) with transfer plasmid and second-generation lentiviral packaging system (psPAX2 and pMD2.G, Addgene #12260 and #12259, gifts from Didier Trono) or pCL-Ampho (Novus) for lentiviral or retroviral production, respectively. Virus was collected 48–72 h post transfection, clarified by 0.45-μm filters. Recipient cells were infected at 50% confluence with virus at a 1:1 dilution with their culturing media containing polybrene (10 μg/mL). Puromycin selection was started 36 h post infection.
9
Quantifying Virus Infectivity in Vero Cells
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Supernatants collected from moDCs were diluted in DMEM supplemented with 1% FBS and used to infect Vero cells for 1hr at 37°C. Cells and inoculum were overlaid with methylcellulose (OptiMEM [Corning], 1% Antibiotic/Antimycotic [Corning], 2% FBS, and 2% methylcellulose [Sigma Aldrich]) and incubated for 72hr at 37°C. Cells were washed with PBS to remove methylcellulose and fixed with a 1:1 methanol:acetone mixture for 30min. Cells were blocked with 5% milk in PBS at RT for 20min. Cells were incubated with primary antibody (mouse 4G2 monoclonal antibody) at 1μg/mL in 5% milk in PBS for 2hr at RT. Cells were incubated with secondary antibody (HRP-conjugated goat anti-mouse IgG) diluted 1:3000 in 5% milk in PBS for 1hr at RT. Foci were developed with TrueBlue Peroxidase Substrate (KPL). Plates were read on a CTL-ImmunoSpot S6 Micro Analyzer.
10
In Vivo Imaging of ERK Inhibition
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For imaging ERK inhibition in subcutaneously
(s.c.) or intramuscularly (i.m.) implanted cells, U87-EGFRvIII cells
stably expressing KiMBIs were dissociated with trypsin and resuspended
in DMEM and then changed to PBS with a density of 1 × 107 to 2 × 107 cells per mL. For s.c. implantation,
4 × 105 or 1 × 106 cells were resuspended
in 100 μL FBS-free Opti-MEM containing 50% Matrigel matrix (Corning).
For i.m. implantation, 8 × 105 cells were directly
injected in DMEM suspension. First, under sterile conditions, 8- to
10-week-old male nude mice (strain J:NU no. 007850, Jackson Laboratories)
were anesthetized using isoflurane. Cells were subcutaneously injected
into the flanks of nude mice. Mice were recovered on heat pads for
30 min while cells were allowed to settle. One week after cell implantation,
for visualizing tumor growth, mice were i.p. injected with 1.5 μmol
(0.5 mg) of AkaLumine-HCl (Sigma-Aldrich) in 100 μL 0.9% NaCl
for imaging (1 × 1 binning, 10 s exposure time). To visualize
ERK inhibition, tumor-bearing mice were i.p. injected with FFz (0.9
μmol, with 6 mg P-407). Immediately afterward, image acquisition
was initiated and then continued for 10–20 min (1 min per image).
Two h after first-round imaging, kinase inhibitors were injected i.p.,
and then 2 h later, second-round imaging was performed following the
same procedures.
(s.c.) or intramuscularly (i.m.) implanted cells, U87-EGFRvIII cells
stably expressing KiMBIs were dissociated with trypsin and resuspended
in DMEM and then changed to PBS with a density of 1 × 107 to 2 × 107 cells per mL. For s.c. implantation,
4 × 105 or 1 × 106 cells were resuspended
in 100 μL FBS-free Opti-MEM containing 50% Matrigel matrix (Corning).
For i.m. implantation, 8 × 105 cells were directly
injected in DMEM suspension. First, under sterile conditions, 8- to
10-week-old male nude mice (strain J:NU no. 007850, Jackson Laboratories)
were anesthetized using isoflurane. Cells were subcutaneously injected
into the flanks of nude mice. Mice were recovered on heat pads for
30 min while cells were allowed to settle. One week after cell implantation,
for visualizing tumor growth, mice were i.p. injected with 1.5 μmol
(0.5 mg) of AkaLumine-HCl (Sigma-Aldrich) in 100 μL 0.9% NaCl
for imaging (1 × 1 binning, 10 s exposure time). To visualize
ERK inhibition, tumor-bearing mice were i.p. injected with FFz (0.9
μmol, with 6 mg P-407). Immediately afterward, image acquisition
was initiated and then continued for 10–20 min (1 min per image).
Two h after first-round imaging, kinase inhibitors were injected i.p.,
and then 2 h later, second-round imaging was performed following the
same procedures.
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