Plasmids were transfected using the Lipofectamine 2000 protocol (ThermoFisher). Cells were seeded at 0.1 × 106 cells per well in 12 well dishes (Corning) and incubated for 24 h at 37 °C. 500–1000 ng of DNA were incubated with 5 µL Lipofectamine 2000 per well for 20 min at room temperature, and then added to individual wells. Cells were then incubated at 37 °C for 24 h before antibiotic selection for 1–2 weeks. shRNA vectors were selected using 1 µg/mL Puromycin while Rab13HA expression vectors were selected using 50–100 mg/mL Zeocin (ThermoFisher). shRNA transfections were confirmed via GFP visualization and Rab13HA expression was confirmed by western blot detection via an α-HA antibody. Rescue experiments transfected Rab13HA cDNA into shRNA vector expressing cells followed by selection with both Puromycin and Zeocin.
12 well dishes
Manufactured by Corning
Sourced in United States
The 12 well dishes are a fundamental piece of lab equipment used for cell culture and various other experimental procedures. These dishes provide a standardized multi-well format to contain and isolate samples or cell cultures. Each dish contains 12 individual wells, allowing for the simultaneous testing or observation of multiple conditions or samples.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
F1804, by Merck Group (1 mentions)
Sc 5546, by Santa Cruz Biotechnology (1 mentions)
Anti goat sc 2020, by Santa Cruz Biotechnology (1 mentions)
Sc 40, by Abcam (1 mentions)
Halt protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Amersham ecl, by Cytiva (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Observer z1, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Genelute mammalian total rna miniprep kit, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Iscript reverse transcription supermix for rt qpcr, by Bio-Rad (1 mentions)
14 protocols using 12 well dishes
1
Transfection and Selection of Rab13 in Cells
2
Osteoclast Protein Lysate Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell protein lysates were harvested from osteoclasts grown in 12 well dishes (Corning) in modified RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% IGEPAL, 0.25% sodium deoxycholate, 1 mM EDTA) supplemented with Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific). Lysates were cleared by centrifugation at 12,000Xg at 4°C. Proteins were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore), subjected to western blotting by standard protocols, and visualized using ECL Prime (G.E. Health Systems). Blots were blocked and incubated at 4°C with primary antibodies diluted in TBS/0.1% Tween-20 plus 3% bovine serum albumin. Primary antibodies used were HDAC7-Abcam (Ab12174) 1:2000 dilution; α-TUBULIN- Santa Cruz (SC-5546) 1:1000 dilution; MYC- Santa Cruz (SC-40) 1:1000 dilution; MITF- Abcam (Ab12039) 1:1000 dilution; ACTIN- Santa Cruz (SC-1616) 1:2,000 dilution; FLAG—Sigma-Aldrich (F1804) 1:5,000 dilution. Horseradish-peroxidase conjugated secondary antibodies were diluted in TBS/0.1% Tween-20 plus 5% nonfat dry milk. Secondary antibodies used were from G.E. Health Systems: Amersham ECL anti-mouse (NA-931) and anti-rabbit (NA-934) at 1:8000, or Santa Cruz: anti-goat (SC-2020) at 1:12,000 dilution.
3
Immunocytochemical Staining of AtT-20 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunocytochemical staining of AtT-20 cells stably expressing rat proSAAS, cells were first plated on polylysine coated coverslips in 12 well dishes (Corning, Corning, NY, USA). Cells were fixed at low confluence using 4% paraformaldehyde in PBS. Following fixation, cells were rinsed repeatedly with phosphate buffer and then permeablized with 0.1% Triton X-100 detergent in PBS. Cells were then blocked using 4% bovine serum albumin (BSA) in PBS for one hour. Primary antibodies to big LEN, little LEN, SAAS, PEN-LEN, or PEN were incubated with cells in BSA/PBS for one hour. Cells were rinsed with PBS and incubated for one hour with anti-chicken and/or anti-rabbit Cy3 and Cy2 conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA). For experiments using directly labeled primary antibodies, incubation was carried out for one hour, cells were rinsed thoroughly with PBS and affixed to microscope slides using Prolong Gold reagent anti-fade reagent containing 4,6- diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA).
4
Neurite Outgrowth Assay with Nogo66
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CGNs were isolated and plated in the presence or absence of Nogo66 and neurite length determined 30 h post-plating, as described [9 (link), 14 (link), 45 (link), 46 (link)]. All care and procedures for mice were performed in accordance with the guidelines and approval of the University of Michigan University Committee on Use and Care of Animals. 12-well dishes (Corning) were coated with 10 μg/ml poly-l -lysine for 4 h then overnight with 2 μg/ml laminin at room temperature, or laminin plus bacterially expressed His-SUMO Nogo66 (0.75 μg/cm2) at 4°C. Postnatal d8 cerebellar granule neurons (CGNs) were nucleofected as described previously with a total of 6 μg DNA [14 (link)]. 6 h post nucleofection, Y-27632 (10 μM, Calbiochem, San Diego, CA, USA), a ROCK 1/2 inhibitor, or CCG-17444 (25 μM of 3347-0032, ChemDiv, San Diego, CA, USA), were added to CGNs in culture media [DMEM (Invitrogen) supplemented with 2% B27 (Invitrogen), and 1% penicillin/streptomycin (Invitrogen)]. Cells were fixed in 3.7% formaldehyde in PBS 24 h post inhibitor addition. Cells were stained with anti-GFP primary antibody [Molecular Probes (Invitrogen) Cat# A11122 RRID:AB_221569] followed by detection with Alexa Fluor 488 goat anti-rabbit (Life Technologies Cat# A11034 RRID:AB_10562715).
5
Immunofluorescence Staining of DKO-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DKO-1 cells were seeded at 1 × 105 cells per well in 12 well dishes (Corning) containing a single glass coverslip (Fisherbrand). Cells were given ~ 24 h to adhere and grow at 37 °C after which cells were washed twice in 1xPBS and fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were permeabilized using 0.1% Triton X-100 for 15 min at room temperature and then incubated with 5% BSA and 5% donkey serum for 1 h at room temperature. Incubation with primary antibodies in 10% donkey serum at 1:100 dilution was overnight at 4 °C. Cells were then washed three times in 1% donkey serum for 10 min at room temperature, followed by incubation with secondary antibodies in 10% donkey serum at 1:100 dilution for 1 h at room temperature in the dark. Cells were then washed with 1% donkey serum in PBS three times for 10 min at room temperature before being mounted (VectaShield), sealed, and imaged on a Zeiss Observer Z.1.
6
Cell Growth Kinetics Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated at 0.1 × 106 cells per well in 12 well dishes (Corning). Individual wells were collected every 24 h following adherence, and cell counts were calculated using the BioRad Cell Counting System (BioRad). Each time point was collected three different times, and the mean was plotted to calculate a relative exponential growth curve (Prism 9).
7
Synthesis and Imaging of Cy3-GUL Fluorescent Probe
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The chemical synthesis of a cyanine dye-incorporating fluorescent probe (Cy3-GUL) is summarized in SI Appendix, Fig. S25 . SI Appendix includes synthetic chemistry experimental details. For the synthesis of the dye intermediates in SI Appendix, Fig. S25 , please see our previous publication (20 (link)). For in vitro imaging, cells were seeded in 12-well dishes (Corning) overnight. Cells were treated with 100 nM Cy3-GUL and 100 ng/mL Hoechst 33342 (Thermo Scientific) for 5, 10, 30, 60, and 120 min. Cells were washed with phosphate buffered saline (PBS) and prepared for live imaging. SI Appendix contains additional imaging condition.
8
Adipogenic Differentiation of Visceral AMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For imaging, cells were seeded at 10K cells/well in 96-well plates (Cell Carrier, Perkin Elmer #6005550) and induced 4 days after seeding. For RNAseq, cells were seeded at 40K cells/well in 12-well dishes (Corning). Before Induction cells were cultured in proliferation medium (Basic medium consisting of DMEM-F12 1% Penicillin - Streptomycin, 33μM Biotin and 17μM Pantothenate supplemented with 0.13μM Insulin, 0.01μg/ml EGF, 0.001μg/ml FGF, 2.5%FCS). Adipogenic differentiation was induced by changing culture medium to induction medium. (Basic medium supplemented with 0.861μM Insulin, 1nM T3, 0.1μM Cortisol, 0.01 mg/ml Transferrin, 1μM Rosiglitazone, 25nM Dexamethasone, 2.5nM IBMX). On day 3 of adipogenic differentiation culture medium was changed to differentiation medium (Basic medium supplemented with 0.861μM Insulin, 1nM T3, 0.1μM Cortisol, 0.01 mg/ml Transferrin). Medium was changed every 3 days. Visceral-derived AMSCs were differentiated by further adding 2% FBS as well as 0.1mM oleic and linoleic acid to the induction and differentiation media. For isoproterenol stimulation experiments, 1uM isoproterenol was added to the differentiation media and cells treated overnight.
9
Proliferation and Colony Formation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To perform the proliferation assay, 1×10 4 cells were plated in 12-well dishes (Corning) per well. Thereafter, cells were cultured in a nutrient-restricted conditions with 10% dialyzed FBS (Sigma-Aldrich) supplement in DMEM (without pyruvate) (Sigma-Aldrich) and after 12 h, each cell group was counted to de ne the initial cell number. Further, the cells treated with or without aspartate (20 mM) were counted at a 24h interval for 48 or 96 h after which the proliferation rate was calculated. For colony formation assay, seed 1,000 cells in each well of a 6-well dish. When visible cell clones appeared, cells were xed using methanol for 15min and thereafter stained with crystal violet (Beyotime) for 10min. Finally, colony counting was performed using ImageJ software [27]
10
Protocol for Adipogenic Differentiation of AMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For imaging, cells were seeded at 10K cells/well in 96-well plates (Cell Carrier, Perkin Elmer #6005550) and induced 4 days after seeding. For RNAseq, cells were seeded at 40K cells/well in 12-well dishes (Corning). Before Induction cells were cultured in proliferation medium (Basic medium consisting of DMEM-F12 1% Penicillin -Streptomycin, 33µM Biotin and 17µM
Pantothenate supplemented with 0.13µM Insulin, 0.01ug/ml EGF, 0.001ug/ml FGF, 2.5%FCS).
Adipogenic differentiation was induced by changing culture medium to induction medium. (Basic medium supplemented with 0.861μM Insulin, 1nM T3, 0.1μM Cortisol, 0.01mg/ml Transferrin, 1μM Rosiglitazone, 25nM Dexamethasone, 2.5nM IBMX). On day 3 of adipogenic differentiation culture medium was changed to differentiation medium (Basic medium supplemented with 0.861μM Insulin, 1nM T3, 0.1μM Cortisol, 0.01mg/ml Transferrin). Medium was changed every 3 days. Visceral-derived AMSCs were differentiated by further adding 2% FBS as well as 0.1mM oleic and linoleic acid to the induction and differentiation media. For isoproterenol stimulation experiments, 1uM isoproterenol was added to the differentiation media and cells treated overnight.
Pantothenate supplemented with 0.13µM Insulin, 0.01ug/ml EGF, 0.001ug/ml FGF, 2.5%FCS).
Adipogenic differentiation was induced by changing culture medium to induction medium. (Basic medium supplemented with 0.861μM Insulin, 1nM T3, 0.1μM Cortisol, 0.01mg/ml Transferrin, 1μM Rosiglitazone, 25nM Dexamethasone, 2.5nM IBMX). On day 3 of adipogenic differentiation culture medium was changed to differentiation medium (Basic medium supplemented with 0.861μM Insulin, 1nM T3, 0.1μM Cortisol, 0.01mg/ml Transferrin). Medium was changed every 3 days. Visceral-derived AMSCs were differentiated by further adding 2% FBS as well as 0.1mM oleic and linoleic acid to the induction and differentiation media. For isoproterenol stimulation experiments, 1uM isoproterenol was added to the differentiation media and cells treated overnight.
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!