monkey  (Sino Biological)

Journal: Life

Article Title: Ice-Cold Temperature Enhances NADPH Oxidase-Dependent Release of Tissue Factor-Bearing Extracellular Vesicles from Human Monocytic Cells

doi: 10.3390/life16050820

Figure Lengend Snippet: Distinct localization of tissue factor and phosphatidylserine on vesicles, with identification of TF-bearing vesicles within the microvesicle size range. ( A ) Suspension of THP-1 cells shown in the plot of side scatter (SSC) versus forward scatter (FSC). Cells were incubated at 4 °C for 24 h, followed by rewarming at 37 °C for 1 h, and then subjected to flow cytometry after staining with anti-CD142 (tissue factor) antibody conjugated with APC and Annexin V–FITC. ( B ) Same as ( A ), shown with a logarithmic FSC scale. Gating strategy, including the identification of CountBright beads and microvesicle-sized particles (MV), was defined as described in . ( C ) APC fluorescence versus FSC from particles in the THP-1 cell suspension derived from the same sample as in ( A , B , E , F ), without staining with anti-CD142 antibody or Annexin V, showing background APC fluorescence. ( D ) FITC fluorescence versus FSC from the same cell suspension as in ( C ), showing background FITC fluorescence. ( E ) APC fluorescence versus FSC from the same cell suspension as in ( A , B ). ( F ) FITC fluorescence versus FSC from the same cell suspension as in ( A , B ). ( G ) FSC–SSC plot obtained from size-calibration beads for microvesicles (Megamix) mixed with 1 vol% CountBright beads, used to define the gate for microvesicle-sized particles (MV). ( H ) FSC–SSC plot obtained from culture medium containing 2 mM CaCl 2 . ( I ) APC fluorescence versus FITC fluorescence from the same cell suspension as in ( A , B , E , F ), with analysis restricted to particles gated as MV. ( J ) FSC–SSC plot of particles in the suspension derived from the same sample as in ( A , B , E , F ) after centrifugation at 2400× g for 1 min. ( K ) The suspension in ( J ) was further centrifuged at 12,000× g for 15 min to sediment vesicles. FSC–SSC plot of particles in the pellet resuspended in filtered culture medium containing 2 mM Ca 2+ and incubated with anti-CD142 antibody conjugated with APC (10 vol%). ( L ) SSC versus APC fluorescence of particles from the same suspension as in ( K ). Particles gated as MV are indicated in blue.

Article Snippet: Monoclonal human CD142 antibody conjugated to APC was from Miltenyi Biotec (Auburn, CA, USA).

Techniques: Suspension, Incubation, Flow Cytometry, Staining, Fluorescence, Derivative Assay, Centrifugation

Journal: ACS Omega

Article Title: SNAP-Tag-Based Antibody–Drug Conjugates Targeting Epidermal Growth Factor Receptor 1, Epidermal Growth Factor Receptor 2, Trophoblast Cell-Surface Antigen 2, and Tissue Factor for Ovarian Cancer Treatment

doi: 10.1021/acsomega.5c10377

Figure Lengend Snippet: Different expression in ovarian cancer cell lines. The expression levels of EGFR, Trop2, TF, and Her2 in ovarian cancer cell lines were determined by flow cytometry. The cells were treated with anti-EGFR (EGFR monoclonal antibody, H11), anti-Trop2 (Trop2 monoclonal antibody, MR54), anti-TF (CD142 antibody), and anti-Her2 (ErbB2 monoclonal antibody, 3B5) antibodies, respectively, followed by incubation with a secondary antibody (Goat anti-Mouse IgG Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647). The cells were permeabilized to determine the Her2 expression according to manufacturer’s instructions.

Article Snippet: Briefly, 4 × 10 5 cells were collected and washed twice with PBS and then stained with anti-EGFR (H11, 0.5 μg, Thermo Fisher Scientific), anti-Trop2 (MR54, 1 μg, Thermo Fisher Scientific), or anti-TF (CD142, 10 μL, Miltenyi Biotec) antibodies in 200 μL of PBS for 30 min on ice.

Techniques: Expressing, Flow Cytometry, Incubation