|Posted On||03/17/2017 3:20 PM|
|Author||Ming Zeng, Xue Zhong|
|Science Writer||Anne Murray|
The fluoresence activated cell sorting (FACS) screen examines the major hematopoietic cell subsets in the blood in response to aluminum hydroxide (alum)-emulsified ovalbumin (OVA) [OVA-alum].
|Reagents and Solutions|
-1.5 mL Eppendorf microfuge tubes
-Mini-collect EDTA coated tubes
-containing 100 ug OVA
Red blood cell lysis buffer
-0.02% sodium azide
1. Inject 200 uL OVA+Alum (containing 100 ug OVA) via intramuscular (i.m.) injection into the G3 mouse. Return animals to housing.
Blood Preparation for FACS analysis
2. Fourteen days post-OVA+Alum immunization collect 100 μL blood in serum separator tubes.
3. Before staining and flow cytometric analysis, up to 75 μL blood is subjected to two rounds of red blood cell lysis
a) Add 1 mL lysis buffer to the blood and mix for 1 minute
b) Dilute the buffer with 3 mL of PBS
c) Centrifuge at 700 x g for 6 minutes and decant the supernatant.
d) Repeat steps a-c
e) Resuspend cell pellet in 50 μL of 1:200 Fc block in FACS buffer and incubate for 30 minutes on ice.
f) Wash the cells with 1 mL of PBS and spin down the cells at 700 x g for 6 minutes at 4°C.
g) Discard the supernatant and resuspend the cells with 50 μL of antibody cocktails including anti-CD3, CD4, CD8a, NK1.1, CD11b, CD11c, F4/80, CD45.2, B220, IgM, IgD, CD43, CD44, CD5 (1:100-1:300 in FACS Buffer; Table 1) and incubate for 30 minutes on ice.
h) Wash the cells twice with 1 mL of PBS and spin down the cells at 700 x g for 6 minutes at 4°C.
i) Resuspend the cells in 400 μL FACS buffer and analyze the cells on the flow cytometer.
Table 1. Antibodies used in the FACS screen