Human umbilical vein endothelial cells (HUVECs) (Lonza) were used to test the effects of serum on tubulogenesis from different genotypic mice. 5x104 HUVECs (between 2–5 passages) were seeded onto growth factor reduced (GFR) Matrigel (BD Bioscience Cat#354230) on 4-well BD Falcon CultureSlides (Cat#354114). Cells were incubated in the presence of media containing 2% mouse serum from different genotypic groups. After 12 hour incubation (37°C, 5% CO₂), newly formed vessels were imaged using an Olympus 1X51 microscope and analyzed for total number of tubes, tube length and number of nodes. Tube length was measured using ImageJ software.
Falcon cultureslides
Manufactured by BD
Sourced in United States
The BD Falcon CultureSlides are sterile, multi-well cell culture slides designed for various laboratory applications. The slides provide a convenient platform for culturing and observing cells in a controlled environment. Each slide features a transparent surface to support cell growth and microscopic examination.
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Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Huvecs, by BD (1 mentions)
1x51 microscope, by Olympus (1 mentions)
Matrigel, by BD (1 mentions)
Huvecs, by Lonza (1 mentions)
Dylight 594 labeled anti digoxigenin antibody, by Vector Laboratories (1 mentions)
Axioskop 2 mot plus, by Zeiss (1 mentions)
Hoecsht 33342, by Thermo Fisher Scientific (1 mentions)
Prolong antifade kit, by Thermo Fisher Scientific (1 mentions)
Rabbit serum, by Vector Laboratories (1 mentions)
Axiocam mr camera, by Zeiss (1 mentions)
Prolong antifade medium, by Thermo Fisher Scientific (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Sc 8008, by Santa Cruz Biotechnology (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Lsm5 pascal confocal laser scanning microscope, by Zeiss (1 mentions)
17 protocols using falcon cultureslides
1
Serum Effects on HUVEC Tubulogenesis
2
EBER-FISH Assay for EBV Detection
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To validate and confirm infection of AGS cells, EBER-FISH was performed using DIG-labeled probes specific for EBERs (PanPath, Budel, Netherlands). Hybridization was performed according to the manufacturer’s instructions. Briefly, inoculated cells were seeded on microscopy chamber slides (BD Falcon CultureSlides, BD Bioscience). At the indicated time points cells were fixed with 4% Roti-Histofix (Carl Roth AG, Arlesheim, Switzerland) for 15 min at room temperature (RT), washed with 1x PBS, rinsed with ddH2O and dehydrated in 100% EtOH. Subsequently, cells were hybridized with the EBER-probes for 2 h at 37°C in a moist environment. Slides were then washed with 1x PBS and incubated with the secondary Dylight594-labeled anti-Digoxigenin antibody (Vector Laboratories, RECATOLAB, S.A., Servion, Switzerland) for 30 min at RT, washed again with 1x PBS and rinsed with ddH2O. After removing most of the remaining liquid, slides were mounted with VECTASHIELD Mounting Medium (Vector Laboratories) containing 4’,6-diamidino-2-phenylindole (DAPI) to counterstain the nucleus. Slides were analyzed using a fluorescence microscope Axioskop 2 MOT plus (Carl Zeiss, Jena, Germany) and images were process with the AxioVision Rel. 4.8.2 software (Carl Zeiss).
3
Immunofluorescence Staining of β-Tubulin
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For immunofluorescence
staining, cells were plated and grown on BD Falcon CultureSlides (BD
Biosciences, San Jose, CA) and treated with BI 6727 as previously
described. The cells were fixed with a 4% paraformaldehyde in PBS
(pH 7.1) for 15 min at room temperature, then blocked with 5% normal
goat serum in 0.3% Triton X-100 for 1 h. After blocking, cells were
incubated overnight in anti-β-tubulin primary antibody in blocking
buffer (1:100, Cell Signaling no. 2128), followed by a secondary incubation
with Alexa Fluor 594 antirabbit IgG antibody in blocking buffer (5
μg/mL, Invitrogen no. A-11037) for 1 h in the dark. The cells
were then counter-stained with Hoecsht 33342 (Invitrogen, Grand Island,
NY) for nuclear staining, and a ProLong antifade kit was applied per
vendor’s protocol (Molecular Probes, Eugene, OR). Slides were
examined under a Nikon Ti microscope using the respective manufacturer’s
suggested filter sets.
staining, cells were plated and grown on BD Falcon CultureSlides (BD
Biosciences, San Jose, CA) and treated with BI 6727 as previously
described. The cells were fixed with a 4% paraformaldehyde in PBS
(pH 7.1) for 15 min at room temperature, then blocked with 5% normal
goat serum in 0.3% Triton X-100 for 1 h. After blocking, cells were
incubated overnight in anti-β-tubulin primary antibody in blocking
buffer (1:100, Cell Signaling no. 2128), followed by a secondary incubation
with Alexa Fluor 594 antirabbit IgG antibody in blocking buffer (5
μg/mL, Invitrogen no. A-11037) for 1 h in the dark. The cells
were then counter-stained with Hoecsht 33342 (Invitrogen, Grand Island,
NY) for nuclear staining, and a ProLong antifade kit was applied per
vendor’s protocol (Molecular Probes, Eugene, OR). Slides were
examined under a Nikon Ti microscope using the respective manufacturer’s
suggested filter sets.
4
Assessing NFκB Nuclear Translocation
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The nuclear translocation of NFκB was assessed using immunofluorescence on T84 cells grown on 4 well glass chamber slides (BD Falcon culture slides, seeding density 322,000 cells per well).
Two days after seeding, cells were treated with 0.64 μg/ml poly-IC for 30 minutes, a period of time determined by preliminary time course experiments. After a pretreatment with triton X100 (Fluka; 0.1% in PBS for 5 min), and then with rabbit serum (Vector; 1.5% in PBS for 20 min), cells were incubated with a monoclonal antibody directed to NFκB p65 (Santa Cruz, SC-8008; 1:300 for 1 h) and then with alexa fluor A488 rabbit anti-mouse antibodies (InVitrogen, A11059; 1:300 for 1 h). Nuclei were stained with Dapi (1:1000, InVitrogen). Sections were mounted using Prolong anti-fade medium (InVitrogen). The fluorescence was observed on a fluorescent microscope equipped with an Apotome slider which eliminated image blurring (Axiovert 200-M, Carl Zeiss). Image processing was performed using AxioCam MR camera and Axiovision software (Zeiss).
Two days after seeding, cells were treated with 0.64 μg/ml poly-IC for 30 minutes, a period of time determined by preliminary time course experiments. After a pretreatment with triton X100 (Fluka; 0.1% in PBS for 5 min), and then with rabbit serum (Vector; 1.5% in PBS for 20 min), cells were incubated with a monoclonal antibody directed to NFκB p65 (Santa Cruz, SC-8008; 1:300 for 1 h) and then with alexa fluor A488 rabbit anti-mouse antibodies (InVitrogen, A11059; 1:300 for 1 h). Nuclei were stained with Dapi (1:1000, InVitrogen). Sections were mounted using Prolong anti-fade medium (InVitrogen). The fluorescence was observed on a fluorescent microscope equipped with an Apotome slider which eliminated image blurring (Axiovert 200-M, Carl Zeiss). Image processing was performed using AxioCam MR camera and Axiovision software (Zeiss).
5
Visualizing Aberrant Metaphase Plates
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To visualize aberrant metaphase plates, HDFLT/hTERT cells at different passages (P16/P20/P24) were seeded at mid-confluence in eight-well BD Falcon™ CultureSlides (7 × 103 cells for well) (BD Bioscience, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Cells were arrested at pro-metaphase with 75 ng/mL of nocodazole (Sigma-Aldrich, Saint Louis, MI, USA) for 17 h plus 30 min of release, and prepared for immunofluorescence analysis as previously described in Pagotto et al. 2018 [6 (link)]. More than 150 images of metaphases were randomly acquired. Primary antibodies: anti alpha-tubulin (ab7291, Abcam, Cambridge Science Park, UK) and human anti-CREST (Antibodies Inc., Davis, CA, USA). Secondary antibodies: cross-adsorbed species-specific fluorescent secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA). DNA was detected using DRAQ5 (Cell Signaling Technology, Danvers, MA, USA). All the images were acquired using a Carl Zeiss LSM5 Pascal confocal laser scanning microscope (Carl Zeiss 63× Plan Neofluar oil-immersion objective, Oberkochen, Germany).
6
Immunofluorescence Imaging of NF-κB Translocation
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Immunofluorescence staining was performed as previously described (54 (link)). MCF-10A and THP1 cells grown on an eight-well BD Falcon culture slides (BD Biosciences) were either left untreated or treated with Pam3CSK4, or the HIV-1 proteins p17, p24, and gp41. The cells were fixed and immune-stained with NF-κBα p65 (1:500) to detect the nuclear translocation of NF-κBα p65. Corresponding Alexa Fluor 488 conjugated IgG (Molecular Probes, Eugene, OR, USA) was used as a secondary antibody. The images were acquired using an inverted laser-scanning confocal microscope (LSM 510, Zeiss, Oberkochen, Germany).
7
Cell Culture Immunostaining Protocol
8
Isolating Drosophila Larval Hemocytes
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Before hemolymph collection, third instar larvae (hml>GFP+LacZ and hml>HA-hipk3M+GFP) grown at 29°C were washed thoroughly with 1× PBS, dried, and placed in glass dissection wells containing 10 μl of 1× PBS. The cuticle of single larvae was carefully punctured ventrally with forceps, and hemolymph was allowed to drain into the well for 30-60 s. The hemolymph was mixed with a pre-wetted pipet, and 1.5 µl of the hemolymph mixture was transferred to a poly-d -lysine-treated eight-well chamber-slide (BD Falcon CultureSlides; product #354108). Five 1.5 µl droplets were plated per larva, after which they were air dried. The dried samples were washed with 4% formaldehyde for 2 min, washed with PBS, and stained with DAPI. For each sample (n=16), five cell counts were performed from images taken at the center of each droplet at 200× magnification, and means of the five cell counts were plotted; values were subjected to Student's unpaired two-tailed t-test.
9
Immunofluorescence Staining of MEF2C
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For in vitro cultures, cells were seeded in BD Falcon CultureSlides (BD Biosciences) and cultured with M-CSF and RANKL. For sorted cells by flow cytometry, cells were immobilized on slide glasses with Cytospin. Cells were fixed with 4% formaldehyde for 10 min and then blocked with 5%FBS/0.3%TritonX-100/PBS for 60 min. Anti-MEF2C antibody (Cell Signaling Technology) was added to cells in 1:400 dilution with 1%BSA/0.3%TritonX-100/PBS and incubated overnight at 4 °C. After washing with PBS, 1:1 000 diluted Alexa Fluor 488 anti-rabbit IgG (Thermo Scientific) or 1:300 diluted Biotinylated anti-rabbit IgG (Vector Laboratories) were incubated in 1%BSA/0.3%TritonX-100/PBS for 60 min at room temperature. Cells incubated with Biotinylated anti-rabbit IgG were stained with 1:500 diluted streptavidin Alexa Fluor 594 for 30 min at room temperature. Cells were mounted with Fluoroshield Mounting Medium and observed using NIKON SMZ25 with CMOS camera (Zyla 5.5, Andor). Fluorescence intensity was evaluated with ImageJ v1.52a.
10
Multilineage Differentiation of MEi Spheroids
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Day 10 spheroids from MEi cells were transferred from ultra low attachment suspension culture and plated onto non-coated BD Falcon center-well IVF dish (BD Biosciences). The spheroid outgrowths were either differentiated under two conditions: with or without differentiation media. The outgrowths were directly differentiated with STEMPRO osteogenic, adipogenic and chondrogenic differentiation kits (Invitrogen, Life Technologies) and analyzed at day 12 and 24. The outgrowths (both directed and spontaneous differentiation) were stained with Toluidine Blue, Alizarin Red and Oil Red, respectively (all Sigma-Aldrich, MO, USA) to confirm mesenchymal trilineage differentiation. Outgrowths without differentiation media were further confirmed by flow cytometry at day 25 for a panel of mesenchymal stromal cell markers.
Monolayer cultures from MEi cells and BM-MSC were seeded for 48 hours at a cell density of 103 cells in 4 well BD Falcon CultureSlides (BD Biosciences) and were induced for 7 days with 100 ng/ml BMP4 (endoderm) (R&D systems, Sweden) or 100 ng/ml Retinoic acid (mesoderm) (Sigma-Aldrich). Cell culture media was refreshed every second day [34] (link).
Monolayer cultures from MEi cells and BM-MSC were seeded for 48 hours at a cell density of 103 cells in 4 well BD Falcon CultureSlides (BD Biosciences) and were induced for 7 days with 100 ng/ml BMP4 (endoderm) (R&D systems, Sweden) or 100 ng/ml Retinoic acid (mesoderm) (Sigma-Aldrich). Cell culture media was refreshed every second day [34] (link).
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