Location

Schewel Hall Room 222

Access Type

Event

Presentation Type

Oral Presentation

Event Website

http://www.lynchburg.edu/academics/red-letter-day/student-scholar-showcase/

Start Date

6-4-2016 9:45 AM

End Date

6-4-2016 10:00 AM

Abstract

The common method of passage for RAW264.7 murine macrophages is to manually scrape the cells from the culture flask. Manual scraping versus trypzinization has never been directly tested. Identical flasks of RAW 264.7 cells are grown in parallel, one passed by scraping, the other by trypsin digestion. Passage is every 3-4 days. Trypsin digestion is done by washing cells with PBS, adding 3 mL of 0.25% Trypsin with 0.53 mM EDTA, then washing cells in DMEM complete to inactivate the trypsin. Cells are counted by hemacytometer and plated at 4x105 cells in 500µl of DMEM for each culture well of a 24-well plate. RAW 264.7 cells are incubated overnight (12-16 hours) at 37°C and 5% CO2. The cells are then washed two times with DMEM complete and treated with concentrations of 0µl, 1µl, 10µl, and 100ng/ml of lipopolysaccharide (from E. coli O55:B5) and returned to the incubator for 24 hours. At 24 hours nitric oxide production is measured by the Greiss reaction. Two series of experiments have examined RAW 264.7 cells from passage 5 – 25 and suggest that cells passed by trypsin have a higher response than cells passed by scraping.

Faculty Mentor

Dr. David O. Freier

Share

COinS
 
Apr 6th, 9:45 AM Apr 6th, 10:00 AM

Nitric Oxide Response of RAW 264.7 Murine Macrophages Maintained in Parallel; Passage by Scraping versus Passage by Trypzinization

Schewel Hall Room 222

The common method of passage for RAW264.7 murine macrophages is to manually scrape the cells from the culture flask. Manual scraping versus trypzinization has never been directly tested. Identical flasks of RAW 264.7 cells are grown in parallel, one passed by scraping, the other by trypsin digestion. Passage is every 3-4 days. Trypsin digestion is done by washing cells with PBS, adding 3 mL of 0.25% Trypsin with 0.53 mM EDTA, then washing cells in DMEM complete to inactivate the trypsin. Cells are counted by hemacytometer and plated at 4x105 cells in 500µl of DMEM for each culture well of a 24-well plate. RAW 264.7 cells are incubated overnight (12-16 hours) at 37°C and 5% CO2. The cells are then washed two times with DMEM complete and treated with concentrations of 0µl, 1µl, 10µl, and 100ng/ml of lipopolysaccharide (from E. coli O55:B5) and returned to the incubator for 24 hours. At 24 hours nitric oxide production is measured by the Greiss reaction. Two series of experiments have examined RAW 264.7 cells from passage 5 – 25 and suggest that cells passed by trypsin have a higher response than cells passed by scraping.

https://digitalshowcase.lynchburg.edu/studentshowcase/2016/Presentations/29