Primary blood vascular endothelial cells (EC) were isolated using magnetic bead-conjugated anti-Pecam-1 antibodies (Cell Biologics; Chicago, IL) and cultured in DMEM containing 10% FBS and PenStrep. Four-chamber slides (Nunc Lab-Tek) were coated with 250 μl of matrigel (CB40234B, BD Biosciences) and allowed to solidify at 37°C for one hour, followed by seeding of 25,000 EC per well. After 24 hours, images acquired using a Nikon TS-100 Microscope equipped with a Flex camera (Nikon). Morphometric analysis of tube-like structures was performed using Pipeline [18 (link)].
Flex camera
Manufactured by Nikon
Sourced in Japan
The Nikon Flex camera is a compact, lightweight, and durable digital camera designed for laboratory and scientific applications. It features a high-resolution sensor, allowing for detailed image capture. The camera is capable of capturing both still images and video footage, providing versatility for various laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Ts100 microscope, by Nikon (2 mentions)
Growth factor reduced matrigel, by BD (2 mentions)
Enzymatic free cell dissociation buffer, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
24 well culture plate, by Merck Group (1 mentions)
Dmem f12 media, by Welgene (1 mentions)
Phase contrast microscope, by Olympus (1 mentions)
Trypsin edta, by Welgene (1 mentions)
3 protocols using flex camera
1
Isolation and Culture of Endothelial Cells
2
RCMVEC Tube Formation on Matrigel
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RCMVECs were washed three times with Dulbecco’s phosphate-buffered saline (DPBS, Invitrogen), lifted using Enzymatic Free Cell Dissociation Buffer (Millipore) for 15 minutes at 37°C, manually disrupted if necessary, collected, centrifuged at 100×g for 5 minutes, and washed twice with DPBS. RCMVECs were resuspended in 1 mL of the appropriate media per treatment group, counted on a Cell Countess, and diluted accordingly. 20,000 RMVECs were added accordingly to each chamber in 1 mL of media on a four-chamber slide (Nunc Lab-Tek). Chambers were coated with 250 μL of Growth Factor Reduced Matrigel (BD Biosciences) under chilled conditions followed by solidification at room temperature prior to RCMVEC addition. After 24 and 48 hours incubation at 37°C on the Matrigel, 4× and 10× magnification images were taken of the RCMVEC tube formation on a Nikon TS-100 Microscope with Flex camera (Nikon).
3
Capillary-like Tube Formation Assay
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Several 24-well culture plates (Sigma-Aldrich) were coated with 100 µL/well of growth factor reduced Matrigel (Becton, Dickinson and Company), and were solidified at 37°C for 1 hour. Before initiation of the tube formation assay, CPC-WT and CPC-KO were washed twice with PBS to remove floating matter and cells, and detached with 0.025% Trypsin-EDTA (Welgene). We have used CPC with or without pre-treatment of serum deprivation culture condition to see the effects of serum deprivation on CPC. Detached CPCs were resuspended with 1 mL serum-containing DMEM/F12 media (Welgene), and counted. 8×104 cells of CPC were plated on growth factor reduced Matrigel coated 24-well culture plates. CPCs were kept in normoxic and hypoxic cultures. We took images of tube formation on a phase-contrast microscope (Olympus Corporation) with a Flex camera (Nikon Corporation, Tokyo, Japan) at the baseline time and 24 hours later.
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