Mice were sacrificed and spleens were removed. The tissues were dissociated, single cell suspensions prepared, and red blood cells were lysed using GEY'S buffer [27 (link)]. The dissociated splenocytes were treated with FC Block (BD Bioscience) and then stained with the following antibodies; CD3, CD4, CD8, CD25, MHCII (clone M5/114.15.1), B220, γδ-TCR (BD Bioscience), and CLIP (15G4, Santa Cruz Biotechnology). For regulatory T cell (Treg) staining we used the eBioscience mouse regulatory T cell staining Kit according to the manufacturer's directions. Live cells were assessed using the Life Technologies LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit according to the manufacturer's directions. Splenocytes were analyzed using a BD FACS Canto II flow cytometer and data were analyzed using FlowJo software (TreeStar Inc.). See Additional file 1 : Figure S1 for gating strategy.
Clip 15g4
Manufactured by Santa Cruz Biotechnology
CLIP (15G4) is a laboratory equipment product offered by Santa Cruz Biotechnology. Its core function is to facilitate cleavage of DNA or RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Mouse regulatory t cell staining kit, by Thermo Fisher Scientific (1 mentions)
γδ tcr, by BD (1 mentions)
Fc block, by BD (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Rat igg2b eb149 10h5, by Thermo Fisher Scientific (1 mentions)
Treg permeabilization buffers, by Thermo Fisher Scientific (1 mentions)
Rmp1 30, by Thermo Fisher Scientific (1 mentions)
Ebioy ae, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
2 protocols using clip 15g4
1
Murine Splenocyte Isolation and Flow Cytometry
2
Multicolor Flow Cytometry Panel
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Antibodies used include: Ii (In-1, 1:100), H2-M (2E5A, 1:50), Rat IgG1 (R3-34, 1:50; all from BD Biosciences); podoplanin (8.1.1, BioLegend, 1:500); CLIP (15G4, Santa Cruz, 1:5); H2-O (ref. 69 (link); Mags.Ob3, Lisa Denzin, 1:400); 10.1.1 (UVA lymphocyte culture centre, 1:1,000); CD45 (30-F11, 1:1,000), CD31 (390, 1:1,000), MHC-II (M5/114.15.2, 1:50 (Fig. 1 ) to 1:500 (Fig. 6 )), CD8 (53-6.7, 1:1,000), CD4 (GK1.5, 1:1,000), Thy1.1 (HIS51, 1:1,000), Thy1.2 (53-2.1, 1:1,000), CD45.1 (A20, 1:500), CD11c (N418, 1:500), CD11b (M1/70, 1:500), CD69 (H1.2F3, 1:500), CD62L (MEL-14, 1:1,000), CD44 (IM7, 1:1,000), CD25 (PC61.5, 1:500), Y-AE (eBioY-Ae, 1:200), PD-1 (RMP1-30, 1:100), LAG-3 (eBioC9B7W, 1:100), Rat IgG2b (eB149/10H5; all from eBioscience). Intracellular staining for PD-1, LAG-3, Ii, H2-M and H2-O was done using the Cytofix/Cytoperm kit (BD Bioscience), and Ki67 (SolA15, 1:500) and FoxP3 (FJK-16s, 1:100) were stained using Treg permeabilization buffers (eBioscience). Annexin V (1:20) was stained using the eBioscience kit. 4,6-Diamidino-2-phenylindole (DAPI; Sigma) or live/dead aqua (Invitrogen, 1:200) were used to distinguish live cells. Cells were acquired on a FACSCanto II (BD Biosciences) and data analysed using FlowJo (Tree Star).
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