Cd45 alexa fluor 700

All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target” format as follows: eFluor 450-CD122, PE-FoxP3, Allophycocyanin-CD4, PerCP-eFluor 710-TNF-α, PE-Cy7-Thy1.1, PE-Cy7-CD62L and PE-Cy7-IFN-γ were from eBioscience (San Diego, CA); V500-CD44, FITC-CD45.1, FITC-TCRβ, PE-CD25, Alexa Fluor 700-CD45.2, Alexa Fluor 700-CD62L, PE-Cy5-CD44, PE-Cy7-CD4 and Allophycocyanin-Cy7-TCRβ were from BD Biosciences (San Diego, CA); PE-Texas Red-CD4 were from Invitrogen (Carlsbad, CA). PE-PBS-57 (analog of α-Galactosylceramide (α-GalCer)) loaded CD1d tetramer was from the NIAID Tetramer Facility. Cells were stained for flow cytometric analysis as previously described (16 (link)). Briefly, live cells are incubated with Fc block (eBioscience) in 2% fetal bovine serum containing PBS, followed by staining with indicated antibodies against surface markers; to stain cytokines, cells were further fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), permeabilized and stained with cytokine antibodies using PBS containing 0.3% saponin (Sigma). Flow data were acquired a on a FC500 (Beckman Coulter, Brea, CA) or LSRII system (BD Biosciences), and analyzed using FlowJo software (Tree Star Inc., OR).

All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target” format as follows: FITC-IL-17A, PE-Foxp3, Allophycocyanin-IFN-γ, Allophycocyanin-Cy7-CD4, PerCP-Cy5.5-CD25, PE-Cy5-ICOS, PerCP-eFluor 710-TNF-α, and PE-Cy7-Thy1a were from eBioscience (San Diego, CA); FITC-CD25, FITC-CD45.1, FITC-Thy1a, PECF594-CD4, PE-CF594-CD8α, Allophycocyanin-ICOS, Alexa Fluor 700-CD45.2, PECy7-CD4 and Allophycocyanin-Cy7-TCRβ were from BD Biosciences (San Diego, CA); Alexa Fluor 610-CD4 and PE-Texas Red-CD8α were from Invitrogen (Carlsbad, CA); Brilliant Violet 421-NRP1, Alexa Fluor 700-CD45.1 and PE-Cy7-CD62L were from Biolegend (San Diego, CA), Foxp3 staining buffer kit was from eBiosciences. To detect cytokines, cells were stimulated with PMA/Ionomycin and analyzed as previously described (33 ).

Mouse intestinal lamina propria cells were isolated using a Lamina
Propria Dissociation Kit (130-097-410) purchased from Miltenyi biotec (Bergisch,
Gladbach, Germany) according to the manufacturer’s protocol. The prepared
cells were then injected into recipient mice intravenously. For flow cytometry,
the prepared cells were then blocked with 1 μg/million cells 2.4G2
(anti-CD16/anti-CD32 ATCC) in 100μl FACS buffer for 5 minutes at
4°C, followed by 1-time wash of FACS buffer to remove residue 2.4G2 and
then incubated in conjugated mAbs Alexa Fluor 700-CD45.2, FITC- CD103, PE-
F4/80, PerCP-cy5.5- CD11b, APC-MHC class II, PE-Cy7- CD11c, and Pacific
blue-CD103 (BD Biosciences). The labeled cells were then washed 3 times in FACS
buffer and then analyzed using a Becton-Dickinson LSR II flow cytometer. Data
were analyzed using FlowJo (TreeStar, Ashland, OR) (Zhang et al., 2014 (link)).