For the phagocytosis assay 5 × 104 iMG progenitors were seeded onto 8-well chamber slide (IB-80827, iBIDI) coated with PDL and matured for 7 days with 50% medium change every other day. Uptake assays were then performed by exchanging 100% media to fresh media supplemented with either 250 ηM Aβ1–42 HyLite Fluor 647 (AS-60493, AnaSpec), mouse brain purified tdTomato-tagged synaptosomes generated in house, FITC-Dextran 4kD (46944, Sigma), FITC-Dextran 150 kD (46946, Sigma), Zymosan beads-568 (Z23374, ThermoFisher) and 75 nM LysoTracker DND-99 (L7528, ThermoFisher) for 100 min. After elapsed time, iMG were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Fixed iMG were subsequently stained with SNL (FL-1301-2, Vector) to visualise the membrane and with 2 µM Hoechst (62249, ThermoFisher) nuclear stain. Images were acquired using Nikon A1R inverted confocal microscope (Nikon) running the NIS elements software using 60 × oil immersion lens. Acquired images were analysed using FIJI and IMARIS, thresholding to WT/Control, marking ROI and quantified either as % Cell Area or uptake (cargo) volume per total cell volume. Statistical significance was assessed using a Kruskal–Wallis test.
Fitc dextran 150 kd
Manufactured by Merck Group
FITC-Dextran 150 kD is a fluorescent dye-labeled polysaccharide with a molecular weight of 150 kilodaltons. It is a versatile tool used in various applications, including cell tracking, permeability studies, and vascular imaging. The FITC (Fluorescein Isothiocyanate) moiety provides a fluorescent label, allowing for the visualization and quantification of the dextran.
Automatically generated - may contain errors
Lab products found in correlation
Zymosan beads 568, by Thermo Fisher Scientific (2 mentions)
Fl 1301 2, by Vector Laboratories (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Ib 80827, by Ibidi (2 mentions)
Fitc dextran 4kd, by Merck Group (2 mentions)
Lysotracker dnd 99, by Thermo Fisher Scientific (2 mentions)
A1r inverted confocal microscope, by Nikon (2 mentions)
Pkh26, by Merck Group (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Cxcl1, by R&D Systems (1 mentions)
Pkh67, by Merck Group (1 mentions)
3 protocols using fitc dextran 150 kd
1
Phagocytic Uptake Assay for Induced Microglia
2
Murine selectin and integrin chimeras
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Murine P- and E-selectin IgM Fc chimeras and control murine CD45-IgM Fc chimera were described previously (Xia et al., 2002 (link); Yago et al., 2010a (link)). Murine ICAM-1 Fc, CXCL1, and TNF were purchased from R&D Systems. Rat anti–murine E-selectin mAb 9A9 and anti–murine P-selectin mAb RB40.34 have been previously described (Labow et al., 1994 (link); Ley et al., 1995 (link)). The following mAbs to murine proteins were purchased from BD: rat anti-integrin αLβ2 (M17/4), rat anti-αMβ2 (M1/70), rat anti–β2 integrin (GAME-46), hamster anti–ICAM-1 (3E2), and biotinylated rat anti–murine Ly6G. Nonbiotinylated rat anti–murine Ly6G mAb was purchased from BioLegend. Murine mAb 8d4, which recognizes human and murine talin1 and talin2, murine fibrinogen, FITC-dextran (150 kD), and PKH26, PKH67, and CellVue Claret fluorescent dyes were purchased from Sigma-Aldrich. Rabbit polyclonal anti–β-actin IgG was purchased from Cell Signaling Technology. Rabbit polyclonal anti–β2 integrin antibody was purchased from Santa Cruz Biotechnology, Inc. Alexa Fluor 488–conjugated donkey anti–rat IgG antibody was purchased from Invitrogen. HRP-conjugated goat anti–murine IgG and goat anti–rabbit IgG were purchased from Thermo Fisher Scientific.
3
Phagocytic Uptake Assay of Alzheimer's-Related Cargoes
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For phagocytosis assay 5 x 10 4 iMG progenitors were seeded onto 8-well chamber slide (IB-80827, iBIDI) coated with PDL and matured for 7 days with 50% medium change every other day. Uptake assays were then performed by exchanging 100% media to fresh media supplemented with either 250 nM Ab 1- 42 HyLite Fluor 647 (AS-60493, AnaSpec), mouse brain puri ed td Tomato-tagged synaptosomes generated in-house, FITC-Dextran 4kD (46944, Sigma), FITC-Dextran 150 kD (46946, Sigma), Zymosan beads-568 (Z23374, ThermoFisher) and 75 nM LysoTracker DND-99 (L7528, ThermoFisher) for 100 min. After elapsed time, iMG were washed with PBS and xed with 4% paraformadehyde for 30 min. Fixed iMG were subsequently stained with SNL (FL-1301-2, Vector) to visualise the membrane and with 2 uM Hoechst (62249, ThermoFisher) nuclear stain. Images were acquired using Nikon A1R inverted confocal microscope (Nikon) running the NIS elements software using 60x oil immersion lens. Acquired images were analysed using FIJI and IMARIS, thresholding to WT/Control, marking ROI and quanti ed either as %Cell Area or uptake (cargo) volume per total cell volume. Statistical signi cance was assessed using a Kruskal-Wallis test.
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