Journal: Frontiers in Immunology

Article Title: Efficient complement-mediated clearance of immunosuppressed T cells by macrophages

doi: 10.3389/fimmu.2023.1183180

Figure Lengend Snippet: Phagocytosis of opsonized small T cells analyzed by imaging flow cytometry. (A, B) Macrophages were labelled with CD18-FITC and T cells were labelled with Celltracker Red (CMTPX). A composition of both channels was created to determine whether T cells were inside or outside the macrophage after 90 min of coculturing. (B, C) Different T cell:macrophage ratios (1:1 up to 12:1) were used to test the phagocytic capability of macrophages.

Article Snippet: Monoclonal CD11b blocking antibody 44a (10 μg/mL, isolated from the supernatant of hybridoma clones, obtained from the American Type Culture Collection, Rockville, MD), monoclonal CD18 blocking antibody IB4 (10 μg/mL, isolated from the supernatant of hybridoma clones, obtained from the American Type Culture Collection, Rockville, MD), monoclonal CD11c blocking antibody CBR-p150/4G1 (10 μg/mL, Invitrogen), and/or polyclonal CR1 blocking antibody (20 μg/mL, kind gift from prof. dr. M.R.

Techniques: Imaging, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Efficient complement-mediated clearance of immunosuppressed T cells by macrophages

doi: 10.3389/fimmu.2023.1183180

Figure Lengend Snippet: Phagocytosis of small T cells by M2 macrophages. (A) Small T cells either unopsonized or opsonized with serum, heat-inactivated serum, serum including complement inhibitor Cp40, or serum including a scrambled peptide control (Scr) cocultured with M-CSF macrophages. (B) Opsonized small T cells in combination with blockage of macrophage receptors; blocking MoAbs 44a against CD11b, CBR-p150/4G1 against CD11c, IB4 against CD18, and CR1 against CD35 cocultured with M-CSF macrophages. Each dot represents data from a single donor. Shown are P-values <0.05. ****P<0.001, ns = not significant, n, 4-11.

Article Snippet: Monoclonal CD11b blocking antibody 44a (10 μg/mL, isolated from the supernatant of hybridoma clones, obtained from the American Type Culture Collection, Rockville, MD), monoclonal CD18 blocking antibody IB4 (10 μg/mL, isolated from the supernatant of hybridoma clones, obtained from the American Type Culture Collection, Rockville, MD), monoclonal CD11c blocking antibody CBR-p150/4G1 (10 μg/mL, Invitrogen), and/or polyclonal CR1 blocking antibody (20 μg/mL, kind gift from prof. dr. M.R.

Techniques: Control, Blocking Assay

Journal: The Journal of Experimental Medicine

Article Title: Integrin α M β 2 -Mediated Cell Migration to Fibrinogen and Its Recognition Peptides

doi:

Figure Lengend Snippet: Migration of α M β 2 WT transfectants but not mock transfectants to Fg. Human 293 cells (5 × 10 5 /well) expressing α M β 2 (WT, black bars) or mock-transfected cells containing only the neomycin resistance plasmid (white bars) were assessed for their ability to migrate to Fg (50 μg/ml, 150 nM), Fn (50 μg/ml), or medium alone placed in the lower wells of transwell plates. Some cells were pretreated with 20 μg/ml anti-β 1 blocking mAb F4611 (A) or 100 nM NIF; M1/70 and 44a, 20 μg/ml α M -blocking mAb; IB4, 20 μg/ml β 2 -blocking mAb; or LM2/1, 20 μg/ml α M -nonblocking mAb (B) for 30 min before addition to the upper wells. Migration was assessed for 22 h at 37°C and migrated cells were fixed, stained, and counted. Migration data are expressed as mean cells/HPF ± SD for five random fields per well (A) or as a percentage of the migration of the WT cells to Fg (B) with duplicate wells in each experiment from three or more experiments. The x-axis indicates the addition to the upper wells containing cells over the addition to the lower wells. ***Medium alone.

Article Snippet: NIF was a gift from Corvas International. mAbs used were as follows: OKM1 (anti-CD11b, IgG 2b ), 44a (anti-CD11b, IgG 1 ), LM2/1 (anti-CD11b, IgG 1 ), M1/70 (anti-CD11b), IB4 (anti-CD18, IgG 2a ), and W6/32 (anti-MHC class I, IgG 1 ), TS2/18 (anti-α L β 2 , IgG 1 ), and TS1/18 (anti-β 2 , IgG 1 ).

Techniques: Migration, Expressing, Transfection, Plasmid Preparation, Blocking Assay, Staining