Clots were formed from 30% plasma, 0.25 µM Alexa Fluor 488 (AF488) fibrinogen (ThermoFisher Scientific, Massachusetts, USA) and 16 µM phospholipids (Rossix, Molndal, Sweden). In some cases, an AF555 labelled monoclonal antibody against cross-linked fibrin (Zedira, Darmstadt, Germany) was incorporated. Clotting was initiated with 0.1 U/ml thrombin (Sigma-Aldrich, St Louis, USA) and 10.6 mM CaCl2 before adding to Ibidi µ-slide VI0.4 chambers (Ibidi GmbH, Gräfelfing, Germany). Patients from the cryo and Fg-C cohort were matched on their fibrinogen concentration in the pre-transfusion sample, so that cohorts were comparable. Representative images are shown in the manuscript. Images were recorded on Zeiss 880 laser scanning confocal microscope with a 63 X 1.40 oil immersion objective using Zeiss Zen 2012 SP1 software (black edition). Images were analysed using FIJI v1.51 and Diameter J plug in.
Alexa fluor 488 af488 fibrinogen
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 (AF488) fibrinogen is a fluorescently labeled protein used in various life science applications. AF488 is a green-fluorescent dye that can be conjugated to fibrinogen, a blood plasma protein involved in blood clotting. This labeled fibrinogen can be used as a tool for studying fibrin formation, platelet function, and other processes involving fibrinogen.
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2 protocols using alexa fluor 488 af488 fibrinogen
1
Fibrin clot formation assay
2
Encapsulation of CAR-T Cells in Fibrin Gels
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Fibrin gels were obtained by mixing 10:1 volume of fibrinogen depleted of plasminogen, von Willebrand factor, and fibronectin FIB3 (Enzyme Research Laboratories, South Bend, IN) and thrombin (1 NIH U ml−1) and HT 1002a (Enzyme Research Laboratories, South Bend, IN) in Hepes buffer (25 mM Hepes, 150 mM NaCl, and 5 mM CaCl2; pH 7.4). All functional assays were assessed using fibrinogen (3 mg/ml) reacted with thrombin (1 U/ml). CAR-T cells (1 × 106 cells) were encapsulated in the fibrin gel, embedded with optimal cutting temperature (OCT), and immersed in liquid nitrogen. The morphology of the F-CAR-T cells was characterized by cryo-SEM (JEOL 7600F, Gatan Alto). The encapsulation of CAR-T cells in fibrin was further characterized by confocal microscopy (Zeiss LSM 710). Alexa Fluor 488 (AF488)–fibrinogen was purchased from Thermo Fisher Scientific.
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