Micropatterning was performed using a deep-UV light technique to normalize cell shape and adhesion area, as previously described (Azioune et al, 2009 (link)). Glass coverslips (square 22 × 22 mm, 1.5, VWR; or round 25 mm, 1.5; Thermo Fisher Scientific) were activated with plasma (Zepto Plasma System, Diener Electronic) for 2 min. After plasma treatment, coverslips were incubated with 0.2 mg/ml PLL(20)-g [3,5]-PEG(2) (SuSoS) in 10 mM Hepes at pH 7.4, for 1 h, at RT. Coverslips were washed three times with water, and left to dry before being placed on a synthetic quartz photomask (Delta Mask), previously activated with deep-UV light (PSD-UV; Novascan Technologies) for 5 min, using 3 μl of Milli-Q water to seal it to the mask. The coverslips were then irradiated through the photomask with the UV lamp for 5 min and left to dry before being incubated with FBN (25 μg/ml; F1141; Sigma-Aldrich), in 100 mM NaHCO3 at pH 8.6, for 30 min, at RT. Whenever possible, 5 μg/ml Alexa Fluor 647–conjugated fibrinogen (Thermo Fisher Scientific) was added to the FBN mix in order to visualize the pattern surfaces. Cells were added to the freshly incubated coverslips and allowed to spread for 15 min, before removing excess cells and new culture medium was added, and cells were left to fully adhere for another 12–16 h.
Alexa fluor 647 conjugated fibrinogen
Manufactured by Thermo Fisher Scientific
Alexa-Fluor 647 conjugated fibrinogen is a fluorescently labeled protein product. Fibrinogen is a glycoprotein found in blood plasma that plays a crucial role in blood clotting. The Alexa-Fluor 647 dye is covalently attached to the fibrinogen molecule, enabling its visualization and detection in biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Merck Group (2 mentions)
Zepto plasma system, by Diener Electronic (1 mentions)
F1141, by Merck Group (1 mentions)
Zen black software, by Zeiss (1 mentions)
Syto41, by Thermo Fisher Scientific (1 mentions)
Axiocam hr camera, by Zeiss (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Plan apochromat 63x 1.40 na, by Zeiss (1 mentions)
Vialtweeter, by Hielscher (1 mentions)
7 protocols using alexa fluor 647 conjugated fibrinogen
1
Deep-UV Micropatterning for Cell Adhesion
2
Fluorescent Fibrin Gel Formation
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Alexa Fluor 647 conjugated fibrinogen
(ThermoFisher) was reconstituted to 3 mg/mL in 0.1 M NaHCO3 according to the manufacturer’s instructions. RBC free fibrin
assays were performed at an Alexafluor 647-fibrinogen concentration
of 1 mg/mL in fibrinogen assay buffer composed of 50 mM Tris pH 7.5,
0.1 M NaCl, and 20 mM CaCl2. The fluorescent fibrinogen
solution was initially plated on a glass bottom 35 mm dish (Mattek
#1.5 glass coverslip) and imaged on the confocal microscope described
inConfocal Microscopy . Thrombin was
added to a final concentration of 0.05 U/μL and imaged 1 min
later to assess the formation of fluorescent fibrin gel.
(ThermoFisher) was reconstituted to 3 mg/mL in 0.1 M NaHCO3 according to the manufacturer’s instructions. RBC free fibrin
assays were performed at an Alexafluor 647-fibrinogen concentration
of 1 mg/mL in fibrinogen assay buffer composed of 50 mM Tris pH 7.5,
0.1 M NaCl, and 20 mM CaCl2. The fluorescent fibrinogen
solution was initially plated on a glass bottom 35 mm dish (Mattek
#1.5 glass coverslip) and imaged on the confocal microscope described
in
added to a final concentration of 0.05 U/μL and imaged 1 min
later to assess the formation of fluorescent fibrin gel.
3
Photolytic Release of Fibrinogen from RBCs
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RBCs were internally loaded with thrombin
and surface modified with C18-Mel/C18-Cbl-BS
as described inhRBC Mel/BS External Loading . A 1% hematocrit suspension of loaded RBCs was made in L-15 media
containing 1 mg/mL Alexa Fluor 647 conjugated fibrinogen (ThermoFisher).
The suspension was loaded into ibidi μ-slides as described inPhotolytic Release of BSA-Alexa Fluor 647 from Loaded
RBCs . The suspensions were allowed to equilibrate in the dark
for 5–10 min before imaging on the confocal microscope described
inConfocal Microscopy. Images were acquired
in a 512 × 512 pixel window with a 60× oil immersion objective
and a dwell time of 10 μs/pixel. Z stacks were acquired at 1.34
μm intervals for a total of ten optical sections spanning 13.4
μm. Stacks were acquired in a time course where one full set
of stacks was acquired, photolysis was performed, and nine more sets
of stacks followed. Photolysis was performed in a 250 × 250 pixel
circular ROI at 80% 515 nm laser power for a total of 6 s.
and surface modified with C18-Mel/C18-Cbl-BS
as described in
containing 1 mg/mL Alexa Fluor 647 conjugated fibrinogen (ThermoFisher).
The suspension was loaded into ibidi μ-slides as described in
RBCs
for 5–10 min before imaging on the confocal microscope described
in
in a 512 × 512 pixel window with a 60× oil immersion objective
and a dwell time of 10 μs/pixel. Z stacks were acquired at 1.34
μm intervals for a total of ten optical sections spanning 13.4
μm. Stacks were acquired in a time course where one full set
of stacks was acquired, photolysis was performed, and nine more sets
of stacks followed. Photolysis was performed in a 250 × 250 pixel
circular ROI at 80% 515 nm laser power for a total of 6 s.
4
Imaging Staphylococcus aureus Biofilm Formation
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S. aureus expressing either the chromosome-integrated fusion Coa:msfGFP or unmodified Coa were grown overnight in BHI and then diluted to OD600 5. Microwells (µ-Slide 8 Well, 80821, IBIDI) were preconditioned with 180 µl BHI supplemented with 50% plasma, 10 µM Syto41 (S11352, Invitrogen), and 0.4 µg/ml Alexa Fluor 647-conjugated fibrinogen (F35200, Invitrogen) by incubating at 37°C for 30 minutes. Then 20 µl OD600 5 cultures were added and incubated for a further 2 hours. The biofilms were imaged with 405 nm, 488 nm, and 639 nm wavelength excitation and a Plan-Apochromat 63x/1.40 NA oil immersion objective in the LSM700 confocal microscope (Zeiss). Images were captured with the Axiocam HR camera (Zeiss) and using the Zen Black software (Zeiss). GFP fluorescence was detected with 488 nm wavelength excitation and 490-600 nm wavelength emission, and Alexa 647-conjugated fibrinogen was detected with 639 nm wavelength excitation and 640-750 nm emission.
5
PDMS Micropost Array Functionalization
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Functionalization of the PDMS micropost array was achieved using micro-contact printing37 ,38 . Briefly, flat, featureless PDMS stamps were generated and immersed in a solution containing both fibronectin (50 μg mL−1; Sigma-Aldrich) and Alexa-Fluor 647 conjugated fibrinogen (25 μg mL−1; Invitrogen, Carlsbad, CA) for 1 hr. PDMS stamps were rinsed with DI water before blown dry using nitrogen gas. Protein-coated PDMS stamps were then placed in conformal contact with the PDMS micropost array pre-treated with UV-ozone (UV-ozone cleaner, Jelight, Irvine, CA) to transfer adhesive proteins from stamps to the tops of PDMS microposts.
6
PDMS Micropost Array Functionalization
7
Bacteria Surface Labeling Optimization
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Bacteria were sonicated (VialTweeter; Hielscher) for 0.5 min to separate any aggregates and incubated fixed in 4% paraformaldehyde for 5 min on ice. The bacteria were thereafter washed with PBS twice (10,000 × g, 2 min). SF370 wildtype was stained with Alexa Fluor 488-conjugated WGA. Bacteria were incubated with IdeS-cleaved Xolair, Ab49, or Alexa Fluor 647-conjugated Fibrinogen (Invitrogen). The antibody samples were stained with fluorescently labelled IgGFab- or IgGFc-specific F(ab’)2 fragments (Alexa Fluor 647-conjugated anti-human IgGFc or IgGFab; Jackson ImmunoResearch Laboratories). Samples were set on glass slides using ProLong Gold Antifade Mountant with No. 1.5 coverslips.
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