Bone Marrow Macrophages: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(→Notes) |
No edit summary |
||
(2 intermediate revisions by one other user not shown) | |||
Line 1: | Line 1: | ||
==Overview== | ==Overview== | ||
Preparation of bone marraow macrophages from mice. | Preparation of bone marraow macrophages from mice. | ||
==Materials== | ==Materials== | ||
Line 9: | Line 9: | ||
==Procedure== | ==Procedure== | ||
Day 0 | '''Day 0''' | ||
#Dissect out mouse femurs and tibias (total of four bones per mouse). | #Dissect out mouse femurs and tibias (total of four bones per mouse). | ||
#Flush the bone marrow from each bone with 5-10 cc of BM20 using a syringe and a 26G needle. Combine the bone marrow from a single mouse into a 50-mL conical tube. Vortex briefly, and bring total volume to 50mL. | #Flush the bone marrow from each bone with 5-10 cc of BM20 using a syringe and a 26G needle. Combine the bone marrow from a single mouse into a 50-mL conical tube. Vortex briefly, and bring total volume to 50mL. | ||
Line 15: | Line 15: | ||
#Incubate at 37°C/5% CO2. | #Incubate at 37°C/5% CO2. | ||
Day 4 | '''Day 4''' | ||
#Add 8-10 mL BM20/ petri dish. Be careful not to slosh the cells at this stage since 20mL BM20 per Petri dish will be quite full. | #Add 8-10 mL BM20/ petri dish. Be careful not to slosh the cells at this stage since 20mL BM20 per Petri dish will be quite full. | ||
Day 7 | '''Day 7''' | ||
#Remove media, add 5 mL cold 0.02% EDTA in PBS (Sigma E8008). Incubate at 4°C for 10 min, then use a cell scraper to remove the cells from the plastic. | #Remove media, add 5 mL cold 0.02% EDTA in PBS (Sigma E8008). Incubate at 4°C for 10 min, then use a cell scraper to remove the cells from the plastic. | ||
#Transfer cells to a 50-mL conical, spin (1k rpm, 10 min), and resuspend in BM10 for counting. | #Transfer cells to a 50-mL conical, spin (1k rpm, 10 min), and resuspend in BM10 for counting. | ||
#Plate the cells in BM10 (generally 1.5 x 105 cells/ well in a 24 well-plate). Allow cells to rest for 3 days. | #Plate the cells in BM10 (generally 1.5 x 105 cells/ well in a 24 well-plate). Allow cells to rest for 3 days. | ||
Day10 | '''Day10''' | ||
#Cells are now considered “rested” and ready for use. | #Cells are now considered “rested” and ready for use. | ||
Line 44: | Line 44: | ||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | ||
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. | <!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. | ||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:Needs attention]] | [[Category:Needs attention]] | ||
[[Category:In vitro]] | [[Category:In vitro]] | ||
Latest revision as of 04:10, 19 February 2009
Overview
Preparation of bone marraow macrophages from mice.
Materials
Procedure
Day 0
- Dissect out mouse femurs and tibias (total of four bones per mouse).
- Flush the bone marrow from each bone with 5-10 cc of BM20 using a syringe and a 26G needle. Combine the bone marrow from a single mouse into a 50-mL conical tube. Vortex briefly, and bring total volume to 50mL.
- Plate cells into non-TC treated 10-cm petri dishes (Fisherbrand Cat #08-757-12), 10mL per Petri dish, 5 Petri dishes/mouse
- Incubate at 37°C/5% CO2.
Day 4
- Add 8-10 mL BM20/ petri dish. Be careful not to slosh the cells at this stage since 20mL BM20 per Petri dish will be quite full.
Day 7
- Remove media, add 5 mL cold 0.02% EDTA in PBS (Sigma E8008). Incubate at 4°C for 10 min, then use a cell scraper to remove the cells from the plastic.
- Transfer cells to a 50-mL conical, spin (1k rpm, 10 min), and resuspend in BM10 for counting.
- Plate the cells in BM10 (generally 1.5 x 105 cells/ well in a 24 well-plate). Allow cells to rest for 3 days.
Day10
- Cells are now considered “rested” and ready for use.
Notes
- Primary macrophages don’t come off TC-treated plastic.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Relevant papers and books
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.