Protocols


Screen (pdf version)
ScreenFACS screen
Posted On03/17/2017 3:20 PM
AuthorMing Zeng, Xue Zhong
Science WriterAnne Murray
Background

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

Materials:

-Syringes

-1.5 mL Eppendorf microfuge tubes

-Mini-collect EDTA coated tubes

-FACs tubes

 

Reagents:

OVA+Alum

-containing 100 ug OVA

 

Red blood cell lysis buffer

 

FACs buffer

-PBS

-1% FCS

-0.02% sodium azide

 

PBS

 

Antibody cocktails

Method

Priming

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

Antibody Readout
anti-CD3 CD3+ T cells
anti-CD4 CD4T cells
anti-CD8a CD8adendritic cells (DCs)
anti-NK1.1 NK1.1+ natural killer cells
anti-CD11b CD11b​macrophages
anti-CD11c CD11c​DCs
anti-F4/80 Macrophages
anti-CD45.2 T-cell and B-cell antiggen receptor signal transduction, cytokine signaling, and Fc receptor stimulation
anti-B220 B cells (pro-B to mature)
anti-IgM B cell memory (humoral immune responses)
anti-IgD B cells
anti-CD43 Bone marrow-derived cells (γ/δ T cells, macrophages, mature B cells, and dendritic cells)
anti-CD44 B and T lymphocytes, monocytes, granulocytes, and red blood cells
anti-CD5 T cells
 
Alleles Identified
chestnut
chives
craw
currant
endive
Hulk
kiwis
Lemon
lentil
mushroom
Orange
screamer
snowcock
swan
Thor
waterfowl