Human Albumin Fraction V was purchased from MP Biomedicals (Aurora, Ohio, USA). Albumin was dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg/mL, and the solution was aliquoted to 0.5 mg/0.5 mL per reaction vial. ADIBO-NHS were dissolved in DMSO (5 mg/125 μL) and added to PBS to reach a volume of 500 µL. NHS-compounds were added to a reaction vial by varying the molar ratio (NHS-compounds:albumin, 1.1:1, 3.4:1, 5.7:1, 8.1:1, 11.4:1, 17.2:1, and 23:1; the seven conjugates were named Rxn R1, Rxn R3, Rxn R6, Rxn R8, Rxn R11, Rxn R17, and Rxn R23, respectively). The reaction vials were incubated at room temperature for 2 h and size exclusion chromatography (PD-10 column) was performed using PBS as the mobile phase. A droplet of each fraction was spotted on a glass microfiber filter (Whatman GF/B) before staining with Coomassie blue solution. Fractions showing blue stained droplets were collected for albumin concentration analysis. UV-Vis spectroscopy was used to quantify albumin concentration (µg/µL) by determining the optical density ratio at 260/280 nm. The albumin-ADIBO conjugate was lyophilized for further experiments.
Human albumin fraction 5
Manufactured by MP Biomedicals
Sourced in United States
Human Albumin Fraction V is a laboratory product that provides a purified source of human serum albumin. It is commonly used as a protein supplement or stabilizer in various cell culture and biochemical applications.
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4 protocols using human albumin fraction 5
1
Albumin-ADIBO Conjugation Protocol
2
Isolation of Human Carotid Plaque Cells
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Human carotid plaques were collected during CEA; the culprit segment (5 mm) was used for histology and embedded in paraffin as described elsewhere48 (link). In brief, culprit segments were fixed in 4% formaldehyde and decalcified in 10% EDTA, pH 7.5. Afterwards, culprit segments were embedded in paraffin. Time between surgical removal and plaque processing did not exceed 10 minutes. The inclusion of a small medial layer in the dissected tissue could not be excluded during the surgical procedure. The remainder of the plaque was washed in RPMI and minced into small pieces with a razor blade. The tissue was then digested in RPMI 1640 containing 2.5 mg ml−1 of collagenase IV (Thermo Fisher Scientific), 0.25 mg ml−1 of DNAse I (Sigma-Aldrich) and 2.5 mg ml−1 of Human Albumin Fraction V (MP Biomedicals) at 37 °C for 30 minutes. In cohort 2, 1 µM flavopiridol (Selleck Chemicals) was added to the digestion mixture. Subsequently, the plaque cell suspension was filtered through a 70-µm cell strainer and washed with RPMI 1640. Cells were kept in RPMI 1640 with 1% FCS until subsequent staining for flow cytometry (cohort 1), feature barcoding and FACS (cohort 2) or cryostored in CryoStor cell cryopreservation medium (Sigma-Aldrich) until further use.
3
Optimized Human Endarterectomy Single-Cell Processing
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Human endarterectomy samples from cohort 1 and cohort 2 were digested into a single-cell suspension following the same protocol. 28 (link) In brief, a section (1-5 mm) of the culprit segment of the lesion was stored at -80 °C for immunopeptidomics. The remainder of the lesion was digested into a single-cell suspension by cutting the tissue into pieces of ≈1 mm 2 , followed by digestion with 2.5 mg/mL collagenase IV (Thermo Fisher Scientific), 0.25 mg/mL DNAse I (Sigma), 2.5 mg/mL human albumin fraction V (MP Biomedicals) in RPMI 1640 for 30 minutes at 37 °C. After digestion, plaque tissue was mashed over a 70-µm strainer to create a single-cell suspension and washed in RMPI 1640. Cells were cryostored in Cryostor cell cryopreservation medium (Sigma-Aldrich) at -80 °C until further use.
4
Single-cell Isolation from Endarterectomy Samples
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Human endarterectomy samples were digested into a singlecell suspension following a previously described protocol. 6 (link) In brief, lesions were digested into a single-cell suspension by cutting the tissue into pieces of ≈1 mm 2 , followed by digestion with 2.5 mg/mL collagenase IV (Thermo Fisher Scientific), 0.25 mg/mL DNAse I (Sigma), 2.5 mg/mL Human Albumin Fraction V (MP Biomedicals) in RPMI 1640 for 30 minutes at 37 °C. After digestion, plaque tissue was mashed over a 70-µm strainer to create a single-cell suspension and washed in RPMI
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