DiFi cells were cultured on 35 mm culture dishes with a 1.5 coverslip (P35G-0.170–14-C, MatTek Corporation). The cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and then extracted for 5 min with 1% Triton X-100 in 4% paraformaldehyde in PBS, as previously described3 (link). The cells were washed three times in PBS and blocked in 10% BSA in PBS. The cells were incubated with primary antibodies diluted in 10% BSA at 4 °C overnight and washed three times with PBS. The secondary Alexa Fluor antibodies (anti-rabbit conjugated to Alexa Fluor-488 and anti-mouse conjugated to Alexa Fluor-568) were prepared in blocking buffer and centrifuged at 10,000g for 10 min before incubation with the cells for 1 h at room temperature. The primary antibodies used were: anti-DPEP1 (1:50; Sigma-Aldrich, HPA012783) and anti-CD63 (1:50; BD, 556019).
P35g 0.170 14 c
Manufactured by MatTek
Sourced in United States
The P35G-0.170-14-C is a lab equipment product manufactured by MatTek. It is a cell culture dish with a glass bottom and a diameter of 35 mm. The glass bottom has a thickness of 0.170 mm and is coated with a material that supports the growth and attachment of cells.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Ab150080, by Abcam (2 mentions)
Pa5 104561, by Thermo Fisher Scientific (2 mentions)
W11261, by Thermo Fisher Scientific (2 mentions)
H 1500, by Vector Laboratories (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Hpa012783, by BD (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexafluor donkey anti goat 488, by Thermo Fisher Scientific (1 mentions)
Normocin, by InvivoGen (1 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
A9539, by Merck Group (1 mentions)
P7629, by Merck Group (1 mentions)
E10521, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica camera (1 mentions)
Lifeact gfp, by Ibidi (1 mentions)
17 protocols using p35g 0.170 14 c
1
Immunofluorescence Staining of DiFi Cells
2
Fluorescent Imaging of Embryonic Stem Cells
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E3.75 PdgfraH2B-GFP/+ positive embryos obtained from intercrossing of PdgfraH2B-GFP/+ and CD-1 were used. Isolated pPrE and pEPI cells were cultured in a PDL-coated small drop of N2B27 with or without 25 ng/ml FGF2 on 35 mm glass-bottom dishes (MatTek; P35G-0.170-14-C) at 37°C and 5% CO2 for 45 minutes. The samples were fixed and stained in the same manner as pERM staining described in the STAR Methods . Primary and secondary antibodies were diluted in PBST (PBS supplemented with 0.1% Tween). For the secondary antibody incubation, cells were first incubated with AlexaFluor donkey anti-goat 488 (Thermo Fisher, 1:1000) followed by incubation with Hoechst 33342 and AlexaFluor647 Phalloidin (Thermo Fisher, 1:200) diluted in PBST for another hour. Cells were then gently washed three times with PBST. The imaging dishes were then filled with STORM buffer (50 mM Tris pH 7.5, 10 mM NaCl, 10% glucose (w/v), 27 mM MEA, 40 μg/mL catalase, 5 U/mL pyranose oxidase and 2 mM cyclooctatetraene) and imaged immediately.
3
COS-7 Cell Maintenance and Transfection
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COS-7 cells were maintained in DMEM (Invitrogen) supplemented with 100 U/ml penicillin streptomycin (Invitrogen), 0.1 mg/ml normocin (InvivoGen) and 10% fetal bovine serum (Atlanta Biologicals) at 37°C and 5% CO2. Cells were seeded at a density of 1 × 105 cells per dish in 35 mm dishes (MatTek P35G-0.170-14-C) and grown for 24 h. For transfections, calcium phosphate was used to transfect 1 μg DNA. The cells were then washed with fresh media after 16 hours and allowed to grow for an additional 24 h. Live cell imaging was done in FluoroBrite DMEM (Thermo Fisher A1896702) within an environmental chamber to maintain 37°C, 5% CO2 and 100% humidity.
4
Embryoid Imaging in Controlled Environment
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Embryoids were mounted in 4% agarose wells (Sigma A9539) on 35-mm glass-bottom dishes (MatTek Corporation P35G-0.170-14-C) covered with N2B27 and maintained in a chamber at 37 °C, 5% CO2 for the whole length of acquisition. Imaging was performed using a Leica DMi8 motorized fluorescence microscope with LED light source (Leica 15024) with a Leica-DFC9000GTC-VSC10184 camera and the Leica application suite X (LAS X).
5
Visualizing Zebrafish Vascular Development
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Zebrafish larvae were embedded in 0.7% low-melting agarose with 0.112 mg ml−1 Tricaine (E10521, Sigma) and 0.003% PTU (P7629, Sigma) in glass bottom dishes (MatTek, P35G-0.170-14-C). Images presented in this study were acquired using a Leica SP8 confocal microscope with × 20 multi-immersion and × 40 water immersion objectives and LAS X software. Images were processed using ImageJ. Vascular branching was quantified using a semi-automated ImageJ pipeline (Supplementary Fig. 1n ). Animal numbers used are indicated in figure legends. For zebrafish mutants more than 100 embryos per genotype were analysed. In morpholino experiments morphologically malformed embryos were excluded from analysis.
6
Live-cell imaging of MDCK cells on collagen-coated PAAm gels
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Madin-Darby Canine Kidney (MDCK) type II G cells were transfected with LifeAct-GFP (ibidi, 60101) using the Amaxa Biosystem Nucleofector II system and transfection kit (Lonza, VCA-1005). The LifeAct-GFP MDCK cells were maintained in low glucose DMEM (Invitrogen, 11885) containing 1 g/l sodium bicarbonate, 1% Penicillin-Streptomycin (PenStrep, ThermoFisher, 15140122), 0.5 mg/ml G418 selection reagent (Sigma-Aldrich, G418-RO Roche), and supplemented with 10% (vol/vol) fetal bovine serum (FBS). 25 kPa PAAm gels patterned with 100 μg/ml collagen I (Gibco, A1048301) mixed with 20 μg/ml Alexa Fluor 568 labeled gelatin were cast into Mattek dishes (14 mm glass, Mattek P35G-0.170-14-C). MDCK cells were trypsinized and seeded on the PAAm gels for 16 hours before imaging experiments. Prior to imaging, the media was replaced to low glucose DMEM with no phenol red (ThermoFisher, 11054001) and supplemented with 1% PenStrep, 10% FBS, and 25 mM HEPES buffer. Cells were imaged on a Leica DMI6000B microscope with heated incubation unit at 5 minute intervals using a 40x air objective, NA = 0.6.
7
Multicolor Imaging of Cellular Components
8
Connexin 43 Phosphorylation in Mirabegron-Treated Cells
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The phUSMC from the CD31+ bead separation were plated on 35 mm glass bottom dishes (P35G-0.170–14-C, MatTek Corporation) and grown to 70–90% confluency. Cells were treated with mirabegron (30 μM) ±15-min preincubation of KB Src 4 (30 μM) for one hour. Cells are then washed three times with 1 × PBS and incubated with 4% paraformaldehyde for 15 min, followed by 0.5% triton-x for 5 min, and 5% BSA blocking buffer for 1 h with three PBS washes between each step. Cells were then stained with primary rabbit anti-phospho Y265 connexin 43 in 1% BSA (1:100, PA5–104561, Thermo Fisher Scientific, US), followed by secondary Alexa Fluor 594 (ab150080, Abcam, US). Wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 (W11261, Thermo Fisher Scientific, US) was used to define cell membrane. DAPI mounting medium (H-1500, Vector Laboratories, US) was used to stain the nucleus of the cells. All images were taken with negative controls (lack of primary antibody) to confirm there was no nonselective binding (data not shown).
9
Lysosome Dynamics Imaging in Cells
10
Structured Illumination Microscopy of RBPs
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HeLa cells were seeded in glass-bottom coverslips (P35G-0.170-14-C, MatTek) and fixed 24 h after with 3.7% formaldehyde solution in PBS, followed by a permeabilization step with 0.5% Triton X-100 in PBS. Indirect immunofluorescence staining of YBX1, IMP1 and PABPC1 was performed using the following primary antibodies: goat anti-IMP1 (E-20, Santa Cruz), rabbit anti-YBX1 (ab12148, Abcam) and mouse anti-PABPC1 (ab6125, Abcam). Coverslips were washed 3x with PBS prior to incubation with Alexa Fluor 488, 568 and 647 conjugated secondary antibodies (Thermo Fisher Scientific) for an hour at RT. After that, coverslips were washed 3× with PBS and mounted in VECTASHIELD mounting media (RI = 1.45). Structured Illumination Microscopy (SIM) was performed using a Zeiss ELYRA PS.1 microscope with a Plan-apochromat 63×/1.4 NA oil objective. Raw SIM images were taken using three rotations and reconstructed using ZEN 2011 software (Zeiss). After reconstruction, channel correction was applied using a channel alignment file created by imaging 0.1 μm TetraSpeck Microspheres (Thermo Fisher Scientific). Images were thresholded to remove diffuse background (honeycombs) caused by stray pollutants or some residual autofluorescence on the SIM reconstructed pictures before contrasting.
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