Cd3 ef450

Manufactured by Thermo Fisher Scientific

The CD3 (ef450) is a laboratory equipment product from Thermo Fisher Scientific. It is designed to detect and measure the presence of CD3, a protein found on the surface of T cells. The core function of the CD3 (ef450) is to enable researchers and scientists to analyze and study T cell populations in various biological samples.

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2 protocols using cd3 ef450

Bone Marrow Stromal Cell Isolation

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BM was isolated at sacrifice and frozen at −80°C in 90% FCS +10% DMSO. was defrosted, washed in 2%FCS/PBS before being strained through a 70μm cell strainer. Whole BM cells from each replicate (n = 3) within a group were incubated with 2.5-5μl of hashtag antibody (anti-mouse-TotalSeq, BioLegend) and lineage depletion antibodies (Miltenyi) for 10 min at 4°C. Subsequently, standard magnetic depletion protocol was followed. Eluted Lineage negative cells from each replicate were mixed per group and stained with Celltrace Calcein Red/Orange (ThermoFisher) (viability) for 15 min at RT and anti-mouse; CD3 (ef450, ThermoFisher), B220 (ef450, ThermoFisher), CD11b (ef450, ThermoFisher), GR1 (ef450, ThermoFisher), Ter119 (ef450, ThermoFisher) for 15 min at 4°C to check purity of lineage depletion. Two populations were sorted (BD FACSAriaII); GFP(Jak2)⁻Lin^-, to enrich for stromal cells, and GFP⁺. The populations were mixed 75%/25% respectively and used for the 10x platform.

Pritchard J.E., Pearce J.E., Snoeren I.A., Fuchs S.N., Götz K., Peisker F., Wagner S., Benabid A., Lutterbach N., Klöker V., Nagai J.S., Hannani M.T., Galyga A.K., Sistemich E., Banjanin B., Flosdorf N., Bindels E., Olschok K., Biaesch K., Chatain N., Bhagwat N., Dunbar A., Sarkis R., Naveiras O., Berres M.L., Koschmieder S., Levine R.L., Costa I.G., Gleitz H.F., Kramann R, & Schneider R.K. (2023). Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms. Cell Reports, 43(1), 113608.

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Multiparametric Flow Cytometry of Murine Immune Cells

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Cells were stimulated for 4 hours at 37°C in RPMI complete containing 1X cell stimulation cocktail (eBioscience) and protein transport inhibitor cocktail (eBioscience). Stimulated cells were harvested and stained for surface markers and viability for 30 min at room temperature. Samples were stained with viability dye APCef780 (ThermoFisher), CD45 evolve655 (ThermoFisher), CD4 BV785 (BioLegend), CD3 eF450 (ThermoFisher), CD8a BV605 (BioLegend), CD335 BV510 (BioLegend) and Gr-1 FITC (eBioscience). Following overnight fixation using the Fixation/Permeabilization solution (eBioscience Foxp3 Staining Buffer Set), cells were permeabilized and stained for cytokines IL-17A AF488 (eBioscience), IL-22 PE (eBioscience) and IFNγ PE (eBioscience). Cells were analyzed using the Aria IIIu cytometer and data was analyzed using the FlowJo software.

Iyer N., Grizotte-Lake M., Duncan K., Gordon S.R., Palmer A.C., Calvin C., Zhong G., Isoherranen N, & Vaishnava S. (2020). Epithelium intrinsic vitamin A signaling co-ordinates pathogen clearance in the gut via IL-18. PLoS Pathogens, 16(4), e1008360.

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