igg rabbit anti sheep red blood cell  (Valiant Co Ltd)

Journal: European journal of cell biology

Article Title: Target lysis by cholesterol extraction is a rate limiting step in the resolution of phagolysosomes.

doi: 10.1016/j.ejcb.2023.151382

Figure Lengend Snippet: Fig. 3. NPC1-NPC2 activity is required for target lysis. Vehicle control and U18666A-treated iBMDMs (A) and RAW 264.7 cells (C) pre-loaded with 10 kDa dextran were challenged with opsonized splenocytes (A) or sheep red blood cells (sRBC) (C). Target lysis is indicated by the access of dextran, delivered from lysosomes, to the internal compartments of the target. In B) lysis of splenocytes is quantified 1 h after phagocytosis. In D) lysis of sRBCs is quantified over time. U18666A (1 µg/mL) was added 30 min after phagocytosis. n = 3, each independent experiment counting > 30 phagosomes per condition. E) Strategy for measuring human RBC (hRBC) lysis. F) A hRBC lysis curve was constructed by treating indicated RBC volumes with water and measuring hemoglobin at 405 nm in the supernatants. The same hRBC volumes were treated with MβCD before measuring hemoglobin release. G) Recombinant NPC2 was produced in CHO-K1 cells. 10 µg of total protein per well was loaded for SDS-PAGE. Western blot was developed with anti-NPC2 antibody. H) hRBC were treated with recombinant NPC2, the non-cholesterol binding version NPC2P120S or PBS for 15 min. Cholesterol in supernatants was quantified using the Amplex Red assay. The means ± SEM are graphed. n = 3, each dot represents the mean of an independent experiment run per triplicate. Only significant differences are indicated. *** p < 0.005 **** p < 0.001.

Article Snippet: Splenocytes were opsonized with 0.4 mg/mL anti-mouse CD47 clone MIAP410 (InVivoMAb, cat. BE0283) and sheep red blood cells (sRBC) with 1 mg/mL anti-sRBC antibodies (Cedarlane, CLAG6840) at 37 oC and shaking (950 rpm), then washed and resuspended in PBS. iBMDM seeded onto 18-mm circular coverslips in 12-well plates were challenged with the opsonized targets.

Techniques: Activity Assay, Lysis, Control, Construct, Recombinant, Produced, SDS Page, Western Blot, Binding Assay, Amplex Red Assay

Journal: European journal of cell biology

Article Title: Target lysis by cholesterol extraction is a rate limiting step in the resolution of phagolysosomes.

doi: 10.1016/j.ejcb.2023.151382

Figure Lengend Snippet: Fig. 4. The extraction of cholesterol by NPC2 is a rate limiting step for target degradation. A) iBMDMs expressing NPC2-mCherry (red) and Lamp1-GFP (cyan). Cells imaged at rest (left) or challenged with opsonized target (right). Phagosomes are marked with white arrows. B-C) iBMDMs expressing NPC2-mCherry (red) and pre- loaded with 10 kDa dextran (cyan) overnight. After chasing 1 h with fresh medium, macrophages were challenged with opsonized sRBC in presence of vehicle control or U18666A. Target permeability is indicated by dextran entry into target. U18666A (1 µg/mL) was added 30 min after phagocytosis. Representative images are shown in B. Lysis of sRBCs is quantified over time in C. n = 3 each independent experiment counting > 30 phagosomes per condition. D-E) iBMDMs or NPC2- mCherry expressing iBMDMs were challenged with opsonized-sRBC for 1–4 h in presence of vehicle control or U18666A. Representative images stained for anti- sRBC (magenta), fluorescent NPC2 (cyan) and F-actin (yellow) and are shown in D. In E, the means ± SEM of target splenocyte volume (µm3) is graphed. n = 3, each independent experiment counting > 30 phagosomes per condition. Only significant differences are indicated. **** p < 0.001.

Article Snippet: Splenocytes were opsonized with 0.4 mg/mL anti-mouse CD47 clone MIAP410 (InVivoMAb, cat. BE0283) and sheep red blood cells (sRBC) with 1 mg/mL anti-sRBC antibodies (Cedarlane, CLAG6840) at 37 oC and shaking (950 rpm), then washed and resuspended in PBS. iBMDM seeded onto 18-mm circular coverslips in 12-well plates were challenged with the opsonized targets.

Techniques: Extraction, Expressing, Control, Permeability, Lysis, Staining

Journal: Cell

Article Title: Transmembrane pickets connect cyto-and pericellular-skeletons forming barriers to receptor engagement

doi: 10.1016/j.cell.2017.12.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-sheep red blood cell (rabbit) , MP Biomedicals , Cat. # 0855806.

Techniques: Recombinant, Binding Assay, Isolation, Enzyme-linked Immunosorbent Assay, Software