The co-immunoprecipitation (Co-IP) and western blot (WB) assays were carried out as described previously (59 (link), 60 (link), 63 (link)–69 (link)). In brief, HEK293T cells were seeded on 10 cm petri-dish (Corning) and incubated to ~80% confluence, then cells were co-transfected with 20 μg of plasmid combinations tagged with Myc, Flag, or HA. At 24 h post-transfection, cells were harvested and lysed on ice with 800–1,000 μl RIPA lysis buffer (Beyotime Biotechnology) for 30 min. After that, cell lysates were divided into two parts, 10% lysates were directly prepared as the lysates sample, and 90% lysates were incubated with the indicated Ab (anti-Flag or anti-HA) or nonspecific control mouse antibody (IgG) at 4°C for 6 to 12 h, then the antibody-containing lysates were incubated with 50 μl slurry of protein A/G PLUS-Agarose (Santa Cruz) at 4°C overnight. The bead complex was washed at least three times with 1 ml of PBS. Finally, cell lysates and bead protein complex were subjected to WB analysis to detect the potential interaction of virus-host proteins. The original WB results were shown in the section of Supplementary Material
10 cm petri dish
Manufactured by Corning
Sourced in United States
The 10 cm petri dish is a laboratory equipment used for the cultivation and observation of microorganisms, cells, or other biological samples. It provides a controlled environment for the growth and study of these specimens.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Six well plate, by Corning (1 mentions)
Petri dish, by Corning (1 mentions)
Dmem medium, by PanEco (1 mentions)
Ultracut e ultramicrotome, by Reichert Technologies (1 mentions)
6 protocols using 10 cm petri dish
1
Co-immunoprecipitation and Western Blot Assays
2
Cell Culture and Transfection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AGS, MGC-803, BGC-823, SGC-7901 cell lines were purchased from the Cell Bank of the Shanghai Institute of Cells, Chinese Academy of Science (Shanghai, China). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco) in 10-cm petri dish (Corning) at 37°C with 5% CO2. Cell transfection was performed using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Briefly, cells were seeded in six-well plates (Corning) and grown to a cell density of 30% and then transfected and cultured at 37°C for a further 48 h, followed by harvesting for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and other experiments.
3
Transient Cytokine Co-Expression in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four millions cells were seeded in a 10 cm petri dish (Corning) and incubated overnight at 37°C with CO2. The following plasmids were used for Lipofectamine-mediated cell transfection: pRS5a-IL23p19 (2 μg per petri dish), pRS5a-EBI3 or pRS5a-EBI3-His10 (3 μg per petri dish). Two days after cell transfection, the supernatants were collected and concentrated with a Centriprep YM-30 tube (Millipore) by a 30-minute centrifugation step at 3000 g and 4°C. The concentrated eluates were subsequently used in co-immunoprecipitations and ELISA studies.
4
Generation of Mesenchymal Stem Cell Spheroids
5
Quantifying Cell Senescence and Colony Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Сells were counted using a hemacytometer, and 200 cells were seeded into a 10-cm Petri dish (Costar, U.S.A.) and grown in DMEM medium (PanEco, Russia) supplemented with 15% FBS (HyClone, U.S.A.) in the presence of 5% СО2. On day 7, cells were fixed with 70% ethanol and stained with 0.1% Methylene Blue. This was followed by quantitative analysis of colonies using a stereomicroscope. The cell count of a colony was rounded up to the nearest bigger 2n number. For example, if the number of cells in a colony was three or four, it was recorded as 4; if it was five to eight – as 8; if 129 to 256 – as 256.
Determination of senescence-associated β-galactosidase activity was performed as described in [21 (link)]. Cells were washed three times with PBS (pH 7.2), fixed with 0.4% glutaraldehyde in PBS (pH 7.2), washed three times for 5 min each with PBS (pH 7.2), and stained in fresh, filtered X-gal solution (1 mg/ml X-gal, 3 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 mM MgCl2, 1% Nonidet-P40 in PBS, pH 6.0) overnight at 37°C.
Determination of senescence-associated β-galactosidase activity was performed as described in [21 (link)]. Cells were washed three times with PBS (pH 7.2), fixed with 0.4% glutaraldehyde in PBS (pH 7.2), washed three times for 5 min each with PBS (pH 7.2), and stained in fresh, filtered X-gal solution (1 mg/ml X-gal, 3 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 mM MgCl2, 1% Nonidet-P40 in PBS, pH 6.0) overnight at 37°C.
6
Cellular Uptake of Doped Co3O4 Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular
uptake of nondoped and PdO-doped Co3O4 nanoparticles
was evaluated using TEM.23 (link) There were 1
× 106 BEAS-2B or RAW 264.7 cells in 10 mL culture
medium seeded in a 10 cm Petri dish (Costar, Corning, NY) overnight
growth at 37 °C in a humidified 5% CO2 atmosphere.
Cells were treated with 50 μg/mL nanoparticles for 6 h. After
treatment, cells were gently washed three times with PBS and fixed
in 5 mL of 2% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS)
for 2 h. Cells were scratched from the plate and collected for postfixation
in 1% OsO4 in PBS. After fixation for 1 h, cells were dehydrated
in a graded ethanol series, treated with propylene oxide, and embedded
in Epon. There were 50–70 nm thick sections sliced using a
Reichert-Jung Ultracut E ultramicrotome and captured on Formvar-coated
copper grids. The sections were stained with uranyl acetate and Reynolds
lead citrate and examined on a JEOL 100 CX transmission electron microscope
at 80 kV in the UCLA BRI Electron Microscopy Core.
uptake of nondoped and PdO-doped Co3O4 nanoparticles
was evaluated using TEM.23 (link) There were 1
× 106 BEAS-2B or RAW 264.7 cells in 10 mL culture
medium seeded in a 10 cm Petri dish (Costar, Corning, NY) overnight
growth at 37 °C in a humidified 5% CO2 atmosphere.
Cells were treated with 50 μg/mL nanoparticles for 6 h. After
treatment, cells were gently washed three times with PBS and fixed
in 5 mL of 2% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS)
for 2 h. Cells were scratched from the plate and collected for postfixation
in 1% OsO4 in PBS. After fixation for 1 h, cells were dehydrated
in a graded ethanol series, treated with propylene oxide, and embedded
in Epon. There were 50–70 nm thick sections sliced using a
Reichert-Jung Ultracut E ultramicrotome and captured on Formvar-coated
copper grids. The sections were stained with uranyl acetate and Reynolds
lead citrate and examined on a JEOL 100 CX transmission electron microscope
at 80 kV in the UCLA BRI Electron Microscopy Core.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!