Human Serum Albumin-FITC

Guidelines (The following recommended experimental protocol is for guidance only and needs to be adjusted according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvent: Anhydrous dimethyl sulfoxide (DMSO).
1.2 Recommended concentration: 10 mg/mL.

2. Working Solution Preparation
2.1 Diluent: Carbonate/bicarbonate buffer (pH 9) or 20 mM sodium buffer (pH 8.5).
2.2 Working concentration: 0.1 μM, 1 μM, 5 μM or 1.25 mg/mL.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample type description:
3.1.1 Adherent cells^[1][2]: THP-1 macrophages, THP-1 foam cells, polarized primary human macrophages (M0, M1, M2), human carotid artery smooth muscle cells, human umbilical vein endothelial cells, HepG2 cells, immortalized hepatic epithelial cells (CRL-4020).
3.1.2 Notes on cell detachment: After staining and fixation, scrape cells from plastic culture plates using a scraper[1]; or detach and transfer cells using trypsin digestion[2].
3.2 Incubation conditions:
3.2.1 Incubate with 0.1 μM, 1 μM, or 5 μM HSA-FITC for 1 h, 3 h (at 37°C in a 5% CO₂, 95% air humidified atmosphere), or for up to 24 h[1].
3.2.2 Incubate with 1-50 mg/L HSA-FITC-MWCNTs for durations ranging from 1 minute to 24 h, depending on experimental design[2].
3.3 Washing steps:
3.3.1 After live/dead staining, wash with 1% BSA first, followed by PBS[1].
3.3.2 After probe incubation and before fixation, wash with PBS[1].
3.3.3 Wash cells 3 times with 1× PBS[2].

4. Control Setup
4.1 Negative controls: Unmodified HSA-FITC, non-radioactively labeled probe blocking, IgG1 isotype control for SR-A1 blocking[1].
4.2 Competition controls: Modified LDL particles (MDA-LDL, oxLDL)[1].
4.3 Inhibition controls: Dextran sulfate, cytochalasin D[1].
4.4 Untreated cell control, non-functionalized MWCNTs-treated cell control, and background control for apoptosis detection[2].
4.5 Positive control (for validating the experimental system)[2]; blank control (to exclude reagent fluorescence interference)[2].

5. Detection and Analysis
5.1 Instrument types: Flow cytometer, confocal microscope, real-time imaging microscope, confocal laser scanning microscope.
5.2 Excitation/emission wavelengths: For FITC, excitation at 488 nm and emission at 525 nm; or excitation at 488 nm and emission at BP 505-550 nm.
5.3 Result analysis:
5.3.1 Changes in fluorescence intensity: Modified HSA-FITC (Mal-HSA-FITC, Aco-HSA-FITC) shows significantly higher fluorescence intensity in macrophages than unmodified HSA-FITC, and the uptake increases with prolonged incubation time and increased probe concentration[1]; Green fluorescence is observed in HepG2 cells that endocytose HSA-FITC-MWCNTs, whereas only minimal or no green fluorescence is detected in CRL-4020 cells under the same conditions[2].
5.3.2 Fluorescence localization: Modified HSA-FITC localizes to granular cytoplasmic structures and colocalizes with the lysosome marker LAMP-2[1]; HSA-FITC-MWCNTs localize to punctate intracellular structures and plasma membrane vesicles in HepG2 cells, and colocalize with Cy3-labeled anti-Gp60 antibody and anti-caveolin-1 antibody[2].
5.3.3 Fluorescence visualization: Green fluorescence emitted by FITC is observable[1]; Green fluorescence from HSA-FITC, red fluorescence from Cy3-labeled antibodies, and blue fluorescence from DAPI (nuclear dye) are observable, and the colocalized regions of green and red fluorescence appear as yellow signals in merged images[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.