10.6084/m9.figshare.c.3737758_D4.v1
Sean Wattegedera
Sean
Wattegedera
Yolanda Corripio-Miyar
Yolanda
Corripio-Miyar
Yvonne Pang
Yvonne
Pang
David Frew
David
Frew
Tom McNeilly
Tom
McNeilly
Javier Palarea-Albaladejo
Javier
Palarea-Albaladejo
Colin McInnes
Colin
McInnes
Jayne Hope
Jayne
Hope
Elizabeth Glass
Elizabeth
Glass
Gary Entrican
Gary
Entrican
MOESM4 of Enhancing the toolbox to study IL-17A in cattle and sheep
Springer Nature
2017
intracellular IL -17A
FITC
cells Forward Scatter Height
intracellular cytokines
K-L
vs Vioblue channel
vs FSC-Area
SSC-H
FSC-H
Table 3
ovine T cell subsets section
MOESM 4
intracellular cytokine staining
APC
WC
cell surface markers
IFN -γ
two-colour cell phenotyping
study IL -17A
4. Gating strategy
PBMC
cell marker
FMO
Quadrant region boundaries
antibody controls
representative animal
cytokine IL -17A I-J
IFN -γ. Activated
T cell subsets
Side Scatter Height
2017-04-08 05:00:00
Journal contribution
https://springernature.figshare.com/articles/journal_contribution/MOESM4_of_Enhancing_the_toolbox_to_study_IL-17A_in_cattle_and_sheep/4832071
Additional file 4. Gating strategy for the identification of activated bovine T cell subsets expressing intracellular IL-17A and IFN-γ. Activated bovine PBMC were stained for viability, cell surface markers and intracellular cytokines according to the protocol outlined in “Expression of intracellular IL-17A and IFN-γ by bovine and ovine T cell subsets section” using antibodies listed in Table 3 and acquired using an LSRFortessa™ cell analyzer (Becton–Dickinson). Cells were gated to eliminate dead cells using the Vioblue Live/Dead® Fixable Dead Cell Stain Kit, Side Scatter Height (SSC-H) vs Vioblue channel (A) and to include only single cells Forward Scatter Height (FSC-H) vs FSC-Area (FSC-A) (B). Gated single cells used for subsequent two-colour cell phenotyping and intracellular cytokine staining (C). Quadrant region boundaries were set based on isotype-matched directly conjugated antibody controls (FITC vs APC/Alexafluor 647 channels, D) and (Phycoerythrin vs APC/Alexafluor 647 channels, E) and fluorescence minus one (FMO) controls for each cell marker (WC-1, F; CD4, G; CD8β, H) and for each cytokine IL-17A, I-J and IFN-γ, K-L). Data are shown for one representative animal of four.