Zombie Violet™ Fixable Viability Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Don’t use Tris buffer as a diluent and be sure that the PBS does not contain any other protein like BSA or FBS	Dilute Zombie Violet™ dye at 1:100-1000 in PBS and add to cells. Incubate the cells at room temperature, in the dark, for 15-30 minutes

Upstream tips
Don’t use Tris buffer as a diluent and be sure that the PBS does not contain any other protein like BSA or FBS
Protocol tips
Dilute Zombie Violet™ dye at 1:100-1000 in PBS and add to cells. Incubate the cells at room temperature, in the dark, for 15-30 minutes

Publication protocol

WT and Clec4e−/− mice were coded and randomized for tMCAO surgery and FACs profiling as described above, and in supplementary methods. Microglia were isolated 24 h post tMCAO. For each profiled replicate, two ipsilesional hemispheres (with cerebellum and brainstem removed) were pooled for microglia isolation. For sham-operated animals, the whole forebrain was used and brains were not pooled. After myelin separation by Percoll gradient centrifugation, 80,000 CD45intermediate, CD11b+ microglial cells were sorted from each sample. Doublets were excluded with FSC-A and FSC-H linearity, and dead cells excluded using Zombie Violet™ Fixable Viability Kit (423113, BioLegend). RNA was isolated as described above. Samples were amplified (GeneChip WT Pico Kit 902623, Affymetrix) and processed with the Mouse 2.0ST Gene Array WT pico assay (902463, Affymetrix) by the Ramaciotti Centre for Genomics, University of New South Wales. The expression data (RMA background corrected, quantile normalized) are hosted by www.stemformatics.org (dataset S4M-6731)35 and is available from GEO (Accession GSE77986). The expression threshold was calculated as the median expression of all antigenomic probesets on the microarray (log2 3.38). Probes that failed to be expressed above detection threshold in the majority of biological replicates in at least one comparison group were removed from the analysis. The R/Bioconductor limma36 package was used to find differentially expressed genes (DEG) at FDR p < 0.01

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Live / Dead assay mammalian cells - mouse microglia using Zombie Violet™ Fixable Viability Kit from BioLegend.

Manufacturer protocol

Download the product protocol from BioLegend for Zombie Violet™ Fixable Viability Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Live / Dead assay mammalian cells - mouse microglia using Zombie Violet™ Fixable Viability Kit from BioLegend. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.