B16-F1 cell line was purchased from American Type Culture Collection, ATCC (CRL-6323, sex:male). B16-F1 derived CYFIP1/2 knockout (KO) cells (clone #3) were as described. B16-F1 cells and derivatives were cultured in Dulbecco′s Modified Eagle′s Medium, DMEM (4.5 g/L glucose; Invitrogen), supplemented with 10% fetal calf serum, FCS (Gibco, Paisley, UK), 2 mM glutamine (Thermo Fisher Scientific, Darmstadt, Germany) and penicillin (50 Units/mL)/streptomycin (50 µg/mL) (Thermo Fisher Scientific, Darmstadt, Germany). B16-F1 cells were routinely transfected in 35 mm dishes (Sarstedt, Nümbrecht, Germany), using 0.5 µg DNA in total and 1 µL JetPrime for controls, and 1 µg DNA in total and 2 µL JetPrime for B16-F1-derived knockout cells. After overnight transfection, cells were plated onto acid-washed, laminin (Sigma-Aldrich, Taufkirchen, Germany)-coated (25 µg/mL) coverslips and allowed to adhere for at least 5 h prior to analysis. For determining protein half–life, cycloheximide (Abcam, Amsterdam, The Netherlands) was added at a concentration 20 µg/mL for the times indicated, and followed by Western Blotting.
35 mm dishes
Manufactured by Sarstedt
Sourced in Germany, France, Canada, United States
35 mm dishes are cell culture vessels designed for culturing cells and performing various laboratory experiments. They provide a standardized and controlled environment for cell growth and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Abcam (1 mentions)
Laminin, by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
B16f1 cell line, by American Type Culture Collection (1 mentions)
Erythropoietin, by R&D Systems (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
M3234, by STEMCELL (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 phalloidin, by Thermo Fisher Scientific (1 mentions)
Axiovert fluorescent microscope, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 protocols using 35 mm dishes
1
B16-F1 Cell Line Knockout Workflow
2
Collagen-Coated HeLa Cell Culture
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Adherent HeLa cells were seeded on collagen type I coatings into 35-mm dishes (Sarstedt, Marnay, France). To prepare collagen coatings, collagen type I solution from rat tail tendons (Corning; Sigma-Aldrich, L’Isle d’Abeau Chesnes, France) solubilized in 0.018 M acetic acid, was deposited on petri dishes surface at a final concentration of 5 μg/cm2. Then, coated substrates were dried overnight under sterile conditions at room temperature, and rinsed with distilled water before cell plating. Wild-type or HSF1:eGFP-stably transfected HeLa cells were then seeded (1.8 × cells/dish) in complete DMEM without red phenol and were grown for 48 h before experiments.
3
Erythropoietin-Induced CFU-E Formation
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Single cell suspensions were prepared from CBA/Pk, Char10C and C57BL/6J spleen and bone marrow in IMDM (Life Technologies, ON, Canada) supplemented with 10% FBS and 100U/mL penicillin/streptomycin (Thermo Scientific, UT, USA). Spleen or bone marrow were harvested from 3 mice per group and pooled. Cells were plated into 1% methylcellulose medium supplemented with 15% FBS, 1% BSA, 10 μg/mL insulin and 200 μg/mL transferrin (M3234, Stem Cell Technologies, BC, Canada). Various concentrations (0–200 mU/mL) of erythropoietin (R&D Systems, Minneapolis, MN, USA) as well as 2 mM of L-glutamine (Life Technologies, ON, Canada) were added to the medium. Cells were plated in 35 mm dishes (Sarstedt, Montreal, Canada) at a density of 4x104 cells/mL for spleen and 2x104 cells/mL for bone marrow. CFU-E were scored after 2.5 days of culture in a 5% CO2 humidified incubator kept at 37°C.
4
Quantifying Cell Morphology via Phalloidin Microscopy
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Morphological analysis was performed using phalloidin fluorescence microscopy. Cells were plated at 8 × 104 on glass coverslips (Ø25 mm) in 35 mm dishes (Sarstedt, Newton, NC, USA). After the treatment, cells were fixed with 4% paraformaldehyde for 20 min at 4°C, washed with PBS, and then permeabilized with Triton X-100 (0.25%, Sigma-Aldrich, Munich, Germany) for 15 min. After blockade in 5% bovine serum albumin (BSA, Sigma-Aldrich, Munich, Germany) actin filaments were stained by incubating cells (30 min, RT) with Alexa Fluor 555 phalloidin (Invitrogen, Carlsbad, CA, USA) at 1 : 50 in PBS. Cells were washed with PBS and counterstained with Hoechst 33342 (5 μg/mL, Life Technologies, Invitrogen, Carlsbad, CA, USA). Cells were coverslipped with Mowiol (Calbiochem, Darmstadt, Germany) and images were acquired using Zeiss Axiovert fluorescent microscope (Zeiss, Jena, Germany). To quantitatively characterize cell morphology we used AxioVision Rel. 4.6 software, which automatically measures the 2D cell surface area. Cells were analyzed in five random areas (138 × 104 μm2) per coverslip, with three coverslips for each group, in three independent cell preparations.
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