Saline:
Article Title: Intestinal Parasites and Tuberculosis in Wayuu Indigenous Communities in La Guajira, Colombia: A One Health Approach
Article Snippet: DNA was extracted using the NucleoSpin DNA Stool Kit (Macherey-Nagel, Düren, Germany). .. Prior to extraction, samples underwent a pretreatment that included three washes with phosphate-buffered saline (PBS) 1X, with centrifugation at 1369× g for 5 min during each wash. DNA was finally eluted in a total volume of 40 μL, and DNA quality and concentration were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Prior to extraction, 40 g of the soil samples underwent three wash cycles with PBS 1X, each followed by centrifugation at 1369× g for 5 min. DNA was extracted from 250 mg of https://doi.org/10.3390/parasitologia6040036 sediment using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA, USA), following the manufacturer’s instructions.
Article Title: Epigallocatechin gallate and theaflavin-3,3′-digallate exhibit distinct cytotoxic and genotoxic thresholds in human lymphocytes: An In Vitro comparative study
Article Snippet: Epigallocatechin gallate (EGCG) and theaflavin-3,3′-digallate (TF3) are major tea polyphenols with recognized antioxidant and chemopreventive properties.. Their concentration-dependent cytotoxic and genotoxic effects on human peripheral blood lymphocytes (PBLs) remain incompletely characterized.. Here, we compared the responses of primary human PBLs to EGCG and TF3 (5–200 μg/mL) using CCK-8, cytokinesis-block micronucleus (CBMN), and alkaline comet assays.
Article Title: Rapid Detection and Quantification of Salmonella in Chicken Carcass Rinse Using Point-of-Use Filtration-Isothermal Diagnostic Test
Article Snippet: .. The effect of sample matrices on the detection of Salmonella with the filtration-isothermal kit was evaluated using CCR, BPW, and 1X Phosphate Buffered Saline (PBS; pH 7.4; Thermo Fisher Scientific, Waltham, MA, USA). ..
Article Title: Engineering vascularized 3D tissues: An optimized seeding protocol for collagen-based scaffolds
Article Snippet: .. Reagents/tools , Cell line and medium • Primary human umbilical vein endothelial cells, HUVECs (ATCC, Manasses, VA, cat # PCS–100–010) • Vascular cell basal medium (ATCC, cat # PCS–100–030) supplemented with endothelial cell growth kit-VEGF (ATCC, No. PCS–100–041) and 1 % penicillin/streptomycin (ThermoFisher, cat # 15,140,122) Equipment • PHCbi CO 2 Incubator • Countess 3 Automated Cell Counter (ThermoFisher Scientific) • NuWind Multi-Application Centrifuge • Leica TCS SP5 confocal microscope Reagents • Dulbecco’s phosphate-buffered saline (DPBS, ThermoFisher Scientific, cat # 14,040,182) • Trypsin-EDTA (ATCC, cat # 30–2101) • Cubic collagen-based Spongostan Dental MS0005 (Ethicon, Johnson & Johnson) • 4 % paraformaldehyde (PFA, Boster, cat # AR1068) • Phosphate buffered saline (PBS, ThermoFisher Scientific, cat # 10,010,023) • 1 % bovine serum albumin (BSA, Biophoretics, cat # BCZ11920.1 ) • Phalloidin (FITC, ThermoFisher Scientific, cat # F432) • 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, cat # D1306) Software • LAS AF software • ImageJ Angiogenesis Analysis • GraphPad Prism 10.5.0 Plasticware • T75 cm 2 cell culture flasks (VWR, cat # 10,062–860) • 15 ml conical centrifuge tubes (Oxford Lab Products, cat # OCT-15B) • 0.65 mL microcentrifuge tubes (VWR, cat # 87,003–290) • 12-well plates (VWR, cat # 10,861–556) • Scalpel handle #3 • Sterile surgical carbon steel blades (P&P Medical Surgical, size 10) • Tweezers • 20 μL sterile pipette tips (Oxford Lab Products, cat # XR-20-SLF) • 200 μL sterile pipette tips (Oxford Lab Products, cat # XR-200-SLF) • 1000 μL sterile pipette tips (Oxford Lab Products, cat # XR-1000-SLF) • 20 μL pipette (Eppendorf, cat # ES-20F) • 200 μL pipette (Eppendorf, cat # ES-200F) • 1000 μL pipette (Eppendorf, cat # ES-1000F) • 5 mL serological pipettes (Oxford Lab Products, cat # OSC-5C) • 10 mL serological pipettes (Oxford Lab Products, cat # OSP-10C). .. Experimental design , Endothelial cells were seeded in a commercial 3D collagen scaffold to achieve a vascular network. Scaffolded cells were dyed with DAPI and FITC to visualize branching, which was further quantified using ImageJ Angiogenesis Analysis. Scaffold saturation, cell density, one-sided and two-sided seeding, and incubation period were altered to determine the optimal method for achieving maximal vascularization and branching..
Extraction:Article Title: Intestinal Parasites and Tuberculosis in Wayuu Indigenous Communities in La Guajira, Colombia: A One Health Approach
Article Snippet: DNA was extracted using the NucleoSpin DNA Stool Kit (Macherey-Nagel, Düren, Germany). .. Prior to extraction, samples underwent a pretreatment that included three washes with phosphate-buffered saline (PBS) 1X, with centrifugation at 1369× g for 5 min during each wash. DNA was finally eluted in a total volume of 40 μL, and DNA quality and concentration were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Prior to extraction, 40 g of the soil samples underwent three wash cycles with PBS 1X, each followed by centrifugation at 1369× g for 5 min. DNA was extracted from 250 mg of https://doi.org/10.3390/parasitologia6040036 sediment using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA, USA), following the manufacturer’s instructions.
Centrifugation:Article Title: Intestinal Parasites and Tuberculosis in Wayuu Indigenous Communities in La Guajira, Colombia: A One Health Approach
Article Snippet: DNA was extracted using the NucleoSpin DNA Stool Kit (Macherey-Nagel, Düren, Germany). .. Prior to extraction, samples underwent a pretreatment that included three washes with phosphate-buffered saline (PBS) 1X, with centrifugation at 1369× g for 5 min during each wash. DNA was finally eluted in a total volume of 40 μL, and DNA quality and concentration were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Prior to extraction, 40 g of the soil samples underwent three wash cycles with PBS 1X, each followed by centrifugation at 1369× g for 5 min. DNA was extracted from 250 mg of https://doi.org/10.3390/parasitologia6040036 sediment using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA, USA), following the manufacturer’s instructions.
Concentration Assay:Article Title: Intestinal Parasites and Tuberculosis in Wayuu Indigenous Communities in La Guajira, Colombia: A One Health Approach
Article Snippet: DNA was extracted using the NucleoSpin DNA Stool Kit (Macherey-Nagel, Düren, Germany). .. Prior to extraction, samples underwent a pretreatment that included three washes with phosphate-buffered saline (PBS) 1X, with centrifugation at 1369× g for 5 min during each wash. DNA was finally eluted in a total volume of 40 μL, and DNA quality and concentration were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Prior to extraction, 40 g of the soil samples underwent three wash cycles with PBS 1X, each followed by centrifugation at 1369× g for 5 min. DNA was extracted from 250 mg of https://doi.org/10.3390/parasitologia6040036 sediment using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA, USA), following the manufacturer’s instructions.
Spectrophotometry:Article Title: Intestinal Parasites and Tuberculosis in Wayuu Indigenous Communities in La Guajira, Colombia: A One Health Approach
Article Snippet: DNA was extracted using the NucleoSpin DNA Stool Kit (Macherey-Nagel, Düren, Germany). .. Prior to extraction, samples underwent a pretreatment that included three washes with phosphate-buffered saline (PBS) 1X, with centrifugation at 1369× g for 5 min during each wash. DNA was finally eluted in a total volume of 40 μL, and DNA quality and concentration were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Prior to extraction, 40 g of the soil samples underwent three wash cycles with PBS 1X, each followed by centrifugation at 1369× g for 5 min. DNA was extracted from 250 mg of https://doi.org/10.3390/parasitologia6040036 sediment using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA, USA), following the manufacturer’s instructions.
Modification:
Filtration:Article Title: Rapid Detection and Quantification of Salmonella in Chicken Carcass Rinse Using Point-of-Use Filtration-Isothermal Diagnostic Test
Article Snippet: .. The effect of sample matrices on the detection of Salmonella with the filtration-isothermal kit was evaluated using CCR, BPW, and 1X Phosphate Buffered Saline (PBS; pH 7.4; Thermo Fisher Scientific, Waltham, MA, USA). ..
Control:Article Title: The cellular response capacity (CRC) as a novel immunomonitoring approach in sepsis
Article Snippet: In the present study, whole blood was collected into standard neutral monovettes (Sarstedt, Nümbrecht, Germany) supplemented with 0.5 IU heparin per ml. .. Subsequently, the monovettes were exposed to either phosphate-buffered saline (PBS) with calcium and magnesium (PBS +/+ , #14080055, Gibco, Thermo Fisher Scientific, Waltham, USA) and the bacterial culture media [buffer control of Escherichia coli ( E. coli ) suspension, hereafter referred to as BuC], viable E. coli bacteria (ATCC line 25922, DSMZ, Braunschweig, Germany), or LPS (100 ng/ml, from E. coli O55:B5, #L2637, Sigma Aldrich, Steinheim, Germany). ..
Suspension:Article Title: The cellular response capacity (CRC) as a novel immunomonitoring approach in sepsis
Article Snippet: In the present study, whole blood was collected into standard neutral monovettes (Sarstedt, Nümbrecht, Germany) supplemented with 0.5 IU heparin per ml. .. Subsequently, the monovettes were exposed to either phosphate-buffered saline (PBS) with calcium and magnesium (PBS +/+ , #14080055, Gibco, Thermo Fisher Scientific, Waltham, USA) and the bacterial culture media [buffer control of Escherichia coli ( E. coli ) suspension, hereafter referred to as BuC], viable E. coli bacteria (ATCC line 25922, DSMZ, Braunschweig, Germany), or LPS (100 ng/ml, from E. coli O55:B5, #L2637, Sigma Aldrich, Steinheim, Germany). ..
Bacteria:Article Title: The cellular response capacity (CRC) as a novel immunomonitoring approach in sepsis
Article Snippet: In the present study, whole blood was collected into standard neutral monovettes (Sarstedt, Nümbrecht, Germany) supplemented with 0.5 IU heparin per ml. .. Subsequently, the monovettes were exposed to either phosphate-buffered saline (PBS) with calcium and magnesium (PBS +/+ , #14080055, Gibco, Thermo Fisher Scientific, Waltham, USA) and the bacterial culture media [buffer control of Escherichia coli ( E. coli ) suspension, hereafter referred to as BuC], viable E. coli bacteria (ATCC line 25922, DSMZ, Braunschweig, Germany), or LPS (100 ng/ml, from E. coli O55:B5, #L2637, Sigma Aldrich, Steinheim, Germany). ..
Microscopy:Article Title: Engineering vascularized 3D tissues: An optimized seeding protocol for collagen-based scaffolds
Article Snippet: .. Reagents/tools , Cell line and medium • Primary human umbilical vein endothelial cells, HUVECs (ATCC, Manasses, VA, cat # PCS–100–010) • Vascular cell basal medium (ATCC, cat # PCS–100–030) supplemented with endothelial cell growth kit-VEGF (ATCC, No. PCS–100–041) and 1 % penicillin/streptomycin (ThermoFisher, cat # 15,140,122) Equipment • PHCbi CO 2 Incubator • Countess 3 Automated Cell Counter (ThermoFisher Scientific) • NuWind Multi-Application Centrifuge • Leica TCS SP5 confocal microscope Reagents • Dulbecco’s phosphate-buffered saline (DPBS, ThermoFisher Scientific, cat # 14,040,182) • Trypsin-EDTA (ATCC, cat # 30–2101) • Cubic collagen-based Spongostan Dental MS0005 (Ethicon, Johnson & Johnson) • 4 % paraformaldehyde (PFA, Boster, cat # AR1068) • Phosphate buffered saline (PBS, ThermoFisher Scientific, cat # 10,010,023) • 1 % bovine serum albumin (BSA, Biophoretics, cat # BCZ11920.1 ) • Phalloidin (FITC, ThermoFisher Scientific, cat # F432) • 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, cat # D1306) Software • LAS AF software • ImageJ Angiogenesis Analysis • GraphPad Prism 10.5.0 Plasticware • T75 cm 2 cell culture flasks (VWR, cat # 10,062–860) • 15 ml conical centrifuge tubes (Oxford Lab Products, cat # OCT-15B) • 0.65 mL microcentrifuge tubes (VWR, cat # 87,003–290) • 12-well plates (VWR, cat # 10,861–556) • Scalpel handle #3 • Sterile surgical carbon steel blades (P&P Medical Surgical, size 10) • Tweezers • 20 μL sterile pipette tips (Oxford Lab Products, cat # XR-20-SLF) • 200 μL sterile pipette tips (Oxford Lab Products, cat # XR-200-SLF) • 1000 μL sterile pipette tips (Oxford Lab Products, cat # XR-1000-SLF) • 20 μL pipette (Eppendorf, cat # ES-20F) • 200 μL pipette (Eppendorf, cat # ES-200F) • 1000 μL pipette (Eppendorf, cat # ES-1000F) • 5 mL serological pipettes (Oxford Lab Products, cat # OSC-5C) • 10 mL serological pipettes (Oxford Lab Products, cat # OSP-10C). .. Experimental design , Endothelial cells were seeded in a commercial 3D collagen scaffold to achieve a vascular network. Scaffolded cells were dyed with DAPI and FITC to visualize branching, which was further quantified using ImageJ Angiogenesis Analysis. Scaffold saturation, cell density, one-sided and two-sided seeding, and incubation period were altered to determine the optimal method for achieving maximal vascularization and branching..
Software:Article Title: Engineering vascularized 3D tissues: An optimized seeding protocol for collagen-based scaffolds
Article Snippet: .. Reagents/tools , Cell line and medium • Primary human umbilical vein endothelial cells, HUVECs (ATCC, Manasses, VA, cat # PCS–100–010) • Vascular cell basal medium (ATCC, cat # PCS–100–030) supplemented with endothelial cell growth kit-VEGF (ATCC, No. PCS–100–041) and 1 % penicillin/streptomycin (ThermoFisher, cat # 15,140,122) Equipment • PHCbi CO 2 Incubator • Countess 3 Automated Cell Counter (ThermoFisher Scientific) • NuWind Multi-Application Centrifuge • Leica TCS SP5 confocal microscope Reagents • Dulbecco’s phosphate-buffered saline (DPBS, ThermoFisher Scientific, cat # 14,040,182) • Trypsin-EDTA (ATCC, cat # 30–2101) • Cubic collagen-based Spongostan Dental MS0005 (Ethicon, Johnson & Johnson) • 4 % paraformaldehyde (PFA, Boster, cat # AR1068) • Phosphate buffered saline (PBS, ThermoFisher Scientific, cat # 10,010,023) • 1 % bovine serum albumin (BSA, Biophoretics, cat # BCZ11920.1 ) • Phalloidin (FITC, ThermoFisher Scientific, cat # F432) • 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, cat # D1306) Software • LAS AF software • ImageJ Angiogenesis Analysis • GraphPad Prism 10.5.0 Plasticware • T75 cm 2 cell culture flasks (VWR, cat # 10,062–860) • 15 ml conical centrifuge tubes (Oxford Lab Products, cat # OCT-15B) • 0.65 mL microcentrifuge tubes (VWR, cat # 87,003–290) • 12-well plates (VWR, cat # 10,861–556) • Scalpel handle #3 • Sterile surgical carbon steel blades (P&P Medical Surgical, size 10) • Tweezers • 20 μL sterile pipette tips (Oxford Lab Products, cat # XR-20-SLF) • 200 μL sterile pipette tips (Oxford Lab Products, cat # XR-200-SLF) • 1000 μL sterile pipette tips (Oxford Lab Products, cat # XR-1000-SLF) • 20 μL pipette (Eppendorf, cat # ES-20F) • 200 μL pipette (Eppendorf, cat # ES-200F) • 1000 μL pipette (Eppendorf, cat # ES-1000F) • 5 mL serological pipettes (Oxford Lab Products, cat # OSC-5C) • 10 mL serological pipettes (Oxford Lab Products, cat # OSP-10C). .. Experimental design , Endothelial cells were seeded in a commercial 3D collagen scaffold to achieve a vascular network. Scaffolded cells were dyed with DAPI and FITC to visualize branching, which was further quantified using ImageJ Angiogenesis Analysis. Scaffold saturation, cell density, one-sided and two-sided seeding, and incubation period were altered to determine the optimal method for achieving maximal vascularization and branching..
Cell Culture:Article Title: Engineering vascularized 3D tissues: An optimized seeding protocol for collagen-based scaffolds
Article Snippet: .. Reagents/tools , Cell line and medium • Primary human umbilical vein endothelial cells, HUVECs (ATCC, Manasses, VA, cat # PCS–100–010) • Vascular cell basal medium (ATCC, cat # PCS–100–030) supplemented with endothelial cell growth kit-VEGF (ATCC, No. PCS–100–041) and 1 % penicillin/streptomycin (ThermoFisher, cat # 15,140,122) Equipment • PHCbi CO 2 Incubator • Countess 3 Automated Cell Counter (ThermoFisher Scientific) • NuWind Multi-Application Centrifuge • Leica TCS SP5 confocal microscope Reagents • Dulbecco’s phosphate-buffered saline (DPBS, ThermoFisher Scientific, cat # 14,040,182) • Trypsin-EDTA (ATCC, cat # 30–2101) • Cubic collagen-based Spongostan Dental MS0005 (Ethicon, Johnson & Johnson) • 4 % paraformaldehyde (PFA, Boster, cat # AR1068) • Phosphate buffered saline (PBS, ThermoFisher Scientific, cat # 10,010,023) • 1 % bovine serum albumin (BSA, Biophoretics, cat # BCZ11920.1 ) • Phalloidin (FITC, ThermoFisher Scientific, cat # F432) • 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, cat # D1306) Software • LAS AF software • ImageJ Angiogenesis Analysis • GraphPad Prism 10.5.0 Plasticware • T75 cm 2 cell culture flasks (VWR, cat # 10,062–860) • 15 ml conical centrifuge tubes (Oxford Lab Products, cat # OCT-15B) • 0.65 mL microcentrifuge tubes (VWR, cat # 87,003–290) • 12-well plates (VWR, cat # 10,861–556) • Scalpel handle #3 • Sterile surgical carbon steel blades (P&P Medical Surgical, size 10) • Tweezers • 20 μL sterile pipette tips (Oxford Lab Products, cat # XR-20-SLF) • 200 μL sterile pipette tips (Oxford Lab Products, cat # XR-200-SLF) • 1000 μL sterile pipette tips (Oxford Lab Products, cat # XR-1000-SLF) • 20 μL pipette (Eppendorf, cat # ES-20F) • 200 μL pipette (Eppendorf, cat # ES-200F) • 1000 μL pipette (Eppendorf, cat # ES-1000F) • 5 mL serological pipettes (Oxford Lab Products, cat # OSC-5C) • 10 mL serological pipettes (Oxford Lab Products, cat # OSP-10C). .. Experimental design , Endothelial cells were seeded in a commercial 3D collagen scaffold to achieve a vascular network. Scaffolded cells were dyed with DAPI and FITC to visualize branching, which was further quantified using ImageJ Angiogenesis Analysis. Scaffold saturation, cell density, one-sided and two-sided seeding, and incubation period were altered to determine the optimal method for achieving maximal vascularization and branching..
Sterility:Article Title: Engineering vascularized 3D tissues: An optimized seeding protocol for collagen-based scaffolds
Article Snippet: .. Reagents/tools , Cell line and medium • Primary human umbilical vein endothelial cells, HUVECs (ATCC, Manasses, VA, cat # PCS–100–010) • Vascular cell basal medium (ATCC, cat # PCS–100–030) supplemented with endothelial cell growth kit-VEGF (ATCC, No. PCS–100–041) and 1 % penicillin/streptomycin (ThermoFisher, cat # 15,140,122) Equipment • PHCbi CO 2 Incubator • Countess 3 Automated Cell Counter (ThermoFisher Scientific) • NuWind Multi-Application Centrifuge • Leica TCS SP5 confocal microscope Reagents • Dulbecco’s phosphate-buffered saline (DPBS, ThermoFisher Scientific, cat # 14,040,182) • Trypsin-EDTA (ATCC, cat # 30–2101) • Cubic collagen-based Spongostan Dental MS0005 (Ethicon, Johnson & Johnson) • 4 % paraformaldehyde (PFA, Boster, cat # AR1068) • Phosphate buffered saline (PBS, ThermoFisher Scientific, cat # 10,010,023) • 1 % bovine serum albumin (BSA, Biophoretics, cat # BCZ11920.1 ) • Phalloidin (FITC, ThermoFisher Scientific, cat # F432) • 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, cat # D1306) Software • LAS AF software • ImageJ Angiogenesis Analysis • GraphPad Prism 10.5.0 Plasticware • T75 cm 2 cell culture flasks (VWR, cat # 10,062–860) • 15 ml conical centrifuge tubes (Oxford Lab Products, cat # OCT-15B) • 0.65 mL microcentrifuge tubes (VWR, cat # 87,003–290) • 12-well plates (VWR, cat # 10,861–556) • Scalpel handle #3 • Sterile surgical carbon steel blades (P&P Medical Surgical, size 10) • Tweezers • 20 μL sterile pipette tips (Oxford Lab Products, cat # XR-20-SLF) • 200 μL sterile pipette tips (Oxford Lab Products, cat # XR-200-SLF) • 1000 μL sterile pipette tips (Oxford Lab Products, cat # XR-1000-SLF) • 20 μL pipette (Eppendorf, cat # ES-20F) • 200 μL pipette (Eppendorf, cat # ES-200F) • 1000 μL pipette (Eppendorf, cat # ES-1000F) • 5 mL serological pipettes (Oxford Lab Products, cat # OSC-5C) • 10 mL serological pipettes (Oxford Lab Products, cat # OSP-10C). .. Experimental design , Endothelial cells were seeded in a commercial 3D collagen scaffold to achieve a vascular network. Scaffolded cells were dyed with DAPI and FITC to visualize branching, which was further quantified using ImageJ Angiogenesis Analysis. Scaffold saturation, cell density, one-sided and two-sided seeding, and incubation period were altered to determine the optimal method for achieving maximal vascularization and branching..
Article Title: Bovine endometrial epithelial cells are able to internalize Escherichia coli in vitro.
Article Snippet: In most dairy cows (80%–90%), the uterus becomes contaminated with bacteria.. Usually, the contamination is eliminated during the process of involution, together with extensive tissue remodeling.. However, in 10%–20% of cows, the infection is not controlled, which may lead to chronic uterine inflammation, such as clinical metritis and endometritis (Sheldon et al. 2006, 2009; LeBlanc 2023).
Transferring:Article Title: Engineering vascularized 3D tissues: An optimized seeding protocol for collagen-based scaffolds
Article Snippet: .. Reagents/tools , Cell line and medium • Primary human umbilical vein endothelial cells, HUVECs (ATCC, Manasses, VA, cat # PCS–100–010) • Vascular cell basal medium (ATCC, cat # PCS–100–030) supplemented with endothelial cell growth kit-VEGF (ATCC, No. PCS–100–041) and 1 % penicillin/streptomycin (ThermoFisher, cat # 15,140,122) Equipment • PHCbi CO 2 Incubator • Countess 3 Automated Cell Counter (ThermoFisher Scientific) • NuWind Multi-Application Centrifuge • Leica TCS SP5 confocal microscope Reagents • Dulbecco’s phosphate-buffered saline (DPBS, ThermoFisher Scientific, cat # 14,040,182) • Trypsin-EDTA (ATCC, cat # 30–2101) • Cubic collagen-based Spongostan Dental MS0005 (Ethicon, Johnson & Johnson) • 4 % paraformaldehyde (PFA, Boster, cat # AR1068) • Phosphate buffered saline (PBS, ThermoFisher Scientific, cat # 10,010,023) • 1 % bovine serum albumin (BSA, Biophoretics, cat # BCZ11920.1 ) • Phalloidin (FITC, ThermoFisher Scientific, cat # F432) • 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, cat # D1306) Software • LAS AF software • ImageJ Angiogenesis Analysis • GraphPad Prism 10.5.0 Plasticware • T75 cm 2 cell culture flasks (VWR, cat # 10,062–860) • 15 ml conical centrifuge tubes (Oxford Lab Products, cat # OCT-15B) • 0.65 mL microcentrifuge tubes (VWR, cat # 87,003–290) • 12-well plates (VWR, cat # 10,861–556) • Scalpel handle #3 • Sterile surgical carbon steel blades (P&P Medical Surgical, size 10) • Tweezers • 20 μL sterile pipette tips (Oxford Lab Products, cat # XR-20-SLF) • 200 μL sterile pipette tips (Oxford Lab Products, cat # XR-200-SLF) • 1000 μL sterile pipette tips (Oxford Lab Products, cat # XR-1000-SLF) • 20 μL pipette (Eppendorf, cat # ES-20F) • 200 μL pipette (Eppendorf, cat # ES-200F) • 1000 μL pipette (Eppendorf, cat # ES-1000F) • 5 mL serological pipettes (Oxford Lab Products, cat # OSC-5C) • 10 mL serological pipettes (Oxford Lab Products, cat # OSP-10C). .. Experimental design , Endothelial cells were seeded in a commercial 3D collagen scaffold to achieve a vascular network. Scaffolded cells were dyed with DAPI and FITC to visualize branching, which was further quantified using ImageJ Angiogenesis Analysis. Scaffold saturation, cell density, one-sided and two-sided seeding, and incubation period were altered to determine the optimal method for achieving maximal vascularization and branching..
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