Lp vortex mixer

We collected 4 mL of fasting peripheral venous blood from all HCC patients before GSM–TACE, 10 days after surgery, and 30 days after surgery using heparin anticoagulation tubes. We then added 100 μL whole blood to a dry blank tube, lysed it with 500 μL OptiLyse C Lysing Solution (Beckman Coulter Diagnostics, Brea, California, US), vortexed the solution, and incubated the lysed blood at room temperature for 15 min to assure lysis was complete. Then we added 2 mL phosphate-buffered saline (PBS), and we vortexed and centrifuged the mixture at 300 g for 5 min. After centrifugation, we aspirated the supernatant, resuspended the cell pellet in 500 μL PBS, added 5 μL each of CD14, CD11b, and HLA-DR antibodies, and mixed the solution low speed for 5 s. Finally, we analyzed the samples on a flow cytometer.
2.2.2 Antibodies and laboratory equipment: To determine MDSC frequency, we performed multicolor fluorescence-activated cell sorting analysis using Beckman Coulter CytExpert software version 1.1. We also used a DxFLEX Flow Cytometer (Beckman Coulter), a Labofuge 400R Centrifuge (Thermo Fisher Scientific, Waltham, Massachusetts, US), a LP Vortex Mixer(Thermo Fisher Waltham, Massachusetts, US), and the following anti-human monoclonal antibodies: CD11b-APC-Alexa Fluor 750, CD14-PC7, anti–HLA-DR-ECD, OptiLyse C solution, and PBS (all Beckman Coulter).

In order to degrade tannins of Hibiscus tea, a purified enzyme from the recently isolated Penicillium commune HS2 (Gene Bank accession number MT084558) was utilized. Following solid-state fermentation of the isolated fungal strain on potato peels, the extracellular enzyme was purified by ammonium sulfate precipitation, followed by gel electrophoresis of Sepharose 4-B as designated by Mostafa (2022) (link). The Hibiscus tea was initially prepared by boiling 2 g of Hibiscus calyx powder in 100 ml of distilled water for 5 min, followed by filtration. Using a vortex mixer (LP vortex mixer from Thermo Fisher Scientific, USA), Hibiscus tea was mixed with purified tannase (53.1 U/ml) and incubated at 45 °C for 3 h (till no more gallic acid was produced). Tea was heated to 100 °C to inactivate tannase before being stored at −20 °C until analysis. All physicochemical properties of treated and untreated tea at the same dilution were compared.