BD Biosciences
a-mouse CD69 BUV615
751593
H1.2F3
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I used this antibody to identify characterize T cells in a large panel which also analyses B cells, macrophages, monocytes, DCs and NK cells. I used this antibody on splenocytes obtained from C57BL/6J mice infected with Plasmodium berghei ANKA, a mouse model to investigate malaria. CD69 in particular may be used to identify T cell activation or differentiation state. Furthermore, it may also be expressed on other cell types than just T cells. This clone/ fluorochrome combination worked well to characterize CD69 expression.
Flow Cytometry
C57BL/6 splenocytes
1:100, 20 min, 4°C, in 50ul, in the dark
Fc Block
Fix/Perm buffer, 20 min, 4°C, in 70ul, in the dark
20 min, 4°C, in the dark, in 50µl Perm/wash
Flow cytometry
The fluorochrome BUV615 is mainly excited by the UV-Laser and should only be used if said laser and the appropriate detector/ filter-set is installed on the instrument. I used this antibody for a panel established on a 5L Cytek Aurora where I was able to detect it. Some spillover is observed in the ~PE-Tx-Red/ CF594/ Dazzle/… emission range. However, as CD69 is not a expressed at a high level, this effect was negligible. Accordingly, CD69 BUV615 occupied a unique space in the emission spectrum and co-expression with other T cell markers (also in the aforementioned PE-tandem emission spectrum) could be measured well. The brightness of BUV615 was sufficient to characterize CD69 expression. At an optimal dilution of 1:100 (for me), the price per staining is also acceptable. I would recommend this antibody if planning to extend a panel in the UV-range. Lastly, the use of Brilliant stain buffer or similar is advisable if other BUV/ BV antibodies are used. In the following, you will find my staining protocol in short. Incubation steps were performed at 4°C in the dark. 1.Count and adjust splenocytes to 1.5E6 cells, plate in a 96-well plate. 2.If required, stain cells with viability dye. Wash. 3. Stain with extracellular master mix for 20 min. Wash 2x. 4. Fixate/ Permeabilize with buffer of choice, depending on application. I used ThermoFisher FoxP3 fixation/permeabilization kit for 20 min. Wash with perm/ Wash 2x. 5. Perform intracellular staining for 20 min. Wash 2x. 6. Resuspend in FACS buffer and acquire as soon as possible. Here, data was acquired on a 5L Cytek Aurora.
N/A
Work great even in a large panel
A bit expensive
Though a bit expensive, it works well, I am even able to detect a specfific population rather than a smear of CD69+ cells.