|
Subset
|
Marker
|
Company
|
Clone
|
Sec Ab
|
Dilution
|
Positive control
|
---|
Multicolor FCM
|
1
|
CD29-Alexa488+
|
Biolegend
|
TS2/16
| |
1:20
|
MNCs
|
MHC-II−
|
Serotec
|
CVS20
|
RPE
|
1:50
|
MNCs
|
7-AAD−
|
Calbiochem
| | | | |
CD44-APC+
|
BD
|
IM7
| |
1:20
|
MNCs
|
2
|
CD105-RPE+
|
Serotec
|
SN6
| |
1:10
|
Endothelial cells
|
7-AAD−
|
Calbiochem
| | | | |
CD90+
|
VMRD
|
DH24A
|
Alexa647
|
1:100
|
MNCs
|
3
|
CD45-Alexa488−
|
Serotec
|
F10-89-4
| |
1:5
|
MNCs
|
CD73+
|
Abcam
|
10f1
|
RPE
|
1:5
|
Lymphocytes
|
7-AAD−
|
Calbiochem
| | | | |
4
|
Monocyte-Alexa488−
|
Serotec
|
MAC387
| |
1:2.5
|
MNCs
|
CD79α-Alexa647−
|
Serotec
|
HM57
| |
1:2.5
|
MNCs
|
Single-color FCM
| |
MHC-I+
|
VMRD
|
PT85A
|
RPE
|
1:66
|
MNCs
|
Secondary Ab
|
1 and 3
|
Sheep anti-mouse IgG-RPE
|
Sigma-Aldrich
| | |
1:20
| |
|
2
|
Goat anti-mouse IgG-Alexa647
|
Invitrogen Corporation
| | |
1:200
| |
Isotype controls
|
1 and 4
|
Mouse IgG1-Alexa488
|
Biolegend
| | |
1:20
| |
|
1-3
|
Mouse IgG1-RPE
|
Biolegend
| | |
1:10
| |
|
1
|
Rat IgG2b-APC
|
Biolegend
| | |
1:20
| |
|
2
|
Mouse IgM
|
BD
| |
Alexa647
|
1:50
| |
|
3
|
Mouse IgG2-Alexa488
|
Biolegend
| | |
1:20
| |
|
4
|
Mouse IgG1-Alexa647
|
Biolegend
| | |
1:100
| |
- 7-AAD, 7-aminoactinomycin D; FCM, fluorescence correlation microscopy; MHC, major histocompatibility complex; MNC, mononuclear cell; Sec Ab, secondary antibody.