Babco, Signet, Sternberger Monoclonals, Senetek, Covance

other

Phagocytosis Detection Kit Red

421938

1 kit

405 USD

421938

Phagocytosis Detection Kit Red

1 kit

405 USD

Human, Mouse

NONAB_CHMPRB

Component:1 vial (15 µL) Phagocytosis Detection Beads (2-8°C, protected from light)1 vial (20 µL) Viability Dye Green (2-8°C, protected from light)1 vial (100 µL) DMSO (room temperature)Note: all unused stock solutions should be divided into single-use aliquots and stored at -20°C after preparation. Avoid repeated freeze-thaw cycyles.Required Materials Not Included:Buffer: Phosphate-buffered saline (PBS), phenol-free media, or HBSSComplete growth media (cell type dependent)Detection/Imaging Guidelines:Phagocytosis Detection BeadsEx/Em = 570/600 nmFluorescence microscope filter set: Texas RedFlow cytometry channel: PE with yellow/green laser excitationViability Dye GreenEx/Em = 490/525 nmFluorescence microscope filter set: FITCFlow cytometry channel: FITCPrepare Reagents:;Bring all three components to room temperature: Phagocytosis Detection Beads, Viability Dye Green, and DMSONote: all unused stock solutions should be divided into single-use aliquots and stored at -20°C after preparation. Avoid repeated freeze-thaw cycyles.;Viability Dye Green: Prepare stock solution of Viability Dye Green by adding 20 µL of DMSO.;Then, prepare the desired amount of Viability Dye Green working solution in preferred complete growth media by adding 5 µL of the stock solution to 2 mL growth media. Mix well.Note: all unused stock solutions can be aliquoted into single-use vials and stored at -20°C, protexted from light.;Phagocytosis Detection Beads: Prepare desired volume of Phagocytosis Detection Beads working solution in complete growth media by adding 4 µL for every 1 mL growth media. Mix well.;Live-Cell Imaging Assay Protocol:;Add 16 µL of Phagocytosis Detection Beads working solution to every 200 µL of your preferred complete media. You may add the Phagocytosis Detection Beads working solution directly into each well of adherent cells in complete media. Cells should be about 80% confluent.Note: Final concentration may need to be optimized by end user for certain cell types and assay conditions. Incubate cells at 37°C for 4 hours.Note: The incubation time may need to be adjusted according to cell type and assay conditions.;Add 16 µL of the Viability Dye Green working solution to every 200 µL media in cell culture wells.Note: Final concentration may need to be optimized by end user for certain cell types and assay conditions.;Optional: include live-cell nuclear counterstain such as DRAQ5™ (Cat. No. 424101).;Incubate cells at 37°C for 30 minutes.;Gently wash cells twice with PBS, phenol-free media or HBSS.;Add sufficient amount of PBS, phenol-free media or HBSS to the wells and proceed immediately to livecell imaging.;Flow Cytometry Assay Protocol:;Resuspend cells (1-2 x 10^6) in 200 µL of PBS, HBSS, or Cell Staining Buffer (Cat. No. 420201).;Add 16 µL of Phagocytosis Detection Beads working solution to the cell suspension. Note: Final concentration may need to be optimized by end user for certain cell types and assay conditions. You may also prepare single color tubes for cell only, PE, and FITC for compensation.;Add 16 µL of Viability Dye Green working solution to the cell suspension.Note: Final concentration may need to be optimized by end user for certain cell types and assay conditions.;Incubate at 37°C for 1 hour.;Wash cells twice with PBS, HBSS or Cell Staining Buffer. After the second wash, gently remove supernatant from the cell pellet.;Resuspend in 200-300 µL of PBS, HBSS, or Cell Staining Buffer;Proceed to flow cytometry analysis using an appropriate filter set.;