Sean Lauber:Culturing of Primary Macrophages

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(New page: This procedure describes how to culture macrophages derived from from the broncho-alveolar lavage. However, this can be applied to any source of cells where you'd like to isolate an adhere...)
Current revision (11:04, 18 July 2013) (view source)
 
Line 1: Line 1:
-
This procedure describes how to culture macrophages derived from from the broncho-alveolar lavage. However, this can be applied to any source of cells where you'd like to isolate an adherent population (like macrophages).
+
This procedure describes how to culture macrophages derived from from the broncho-alveolar or peritoneal lavage. However, this can be applied to any source of cells where you'd like to isolate an adherent population (like macrophages).
-
1. Count the cells in your BAL and bring them down to an appropriate concentration for plating using 10% FBS/DMEM (or similar). For 6 well plates I plate 1,000,000 cells (ie for 9.6 cm^2) which is sufficient for DNA extraction afterwards. For RNA extraction you may want to use 2.5 million.
+
1. Count the cells and bring them down to an appropriate concentration for plating using 10% FBS/DMEM (or similar). For 6 well plates I plate 1,000,000 cells (ie for 9.6 cm^2) which is sufficient for DNA extraction afterwards. For RNA extraction you may want to use 2.5 million.  
2. After 2 hours, a portion of the cells will adhere to the plate. This is the adherent population and is mostly (~90%) macrophages. Wash the plate 3 times with PBS.
2. After 2 hours, a portion of the cells will adhere to the plate. This is the adherent population and is mostly (~90%) macrophages. Wash the plate 3 times with PBS.
3. Add warmed 10% FBS/DMEM and allow the cells to settle overnight before working with them.
3. Add warmed 10% FBS/DMEM and allow the cells to settle overnight before working with them.

Current revision

This procedure describes how to culture macrophages derived from from the broncho-alveolar or peritoneal lavage. However, this can be applied to any source of cells where you'd like to isolate an adherent population (like macrophages).

1. Count the cells and bring them down to an appropriate concentration for plating using 10% FBS/DMEM (or similar). For 6 well plates I plate 1,000,000 cells (ie for 9.6 cm^2) which is sufficient for DNA extraction afterwards. For RNA extraction you may want to use 2.5 million.

2. After 2 hours, a portion of the cells will adhere to the plate. This is the adherent population and is mostly (~90%) macrophages. Wash the plate 3 times with PBS.

3. Add warmed 10% FBS/DMEM and allow the cells to settle overnight before working with them.

Personal tools