Calculate the amount needed to thaw all samples, and prepare a separate aliquot of warm media with 1:10000 benzonase (25 U/mL). Each sample will require 20 mL of media with benzonase. If using a 300 uL plate, do an additional wash step (resuspension and centrifugation) at each point.Ģ.17. 96-well plate 300 uL well volume (250 uL washes) or 1.2 mL well volume (500 uL washes). Saponin-based permeabilization buffer: 10x (eg, eBiosciences ca# 00-8333-56)Ģ.13.
Ir-intercalator stock solution from Fluidigm (Rh103-intercalator can be used).Ģ.11. Live-dead stain: 5 mg/mL maleimide-DOTA (eg, Macrocyclics B-272) loaded with 139-Lanthanum* or 115-Indium* (*: natural-abundance metal chloride salt used >95% specified isotope trace-metal pure 99.99%)Ģ.10. Phenotyping antibodies, filtered with 0.1 um spin filtersĢ.9. MilliQ water: no contact with beakers or bottles washed with soap (due to barium content of most commercial soaps).Ģ.8. CyFACS buffer (1x CyPBS with 0.1% BSA, 2 mM EDTA and 0.05% Na Azide made in MilliQ water). CyPBS (1x PBS without heavy metal contaminants, such as 10X PBS from Rockland made in MilliQ water)Ģ.6. 2% Para-formaldehyde (PFA), made in 1x CyPBSĢ.5.
Complete RPMI (RPMI with 10% FBS, P/S, glutamine)Ģ.4. In order for a cell to be seen, a metal in the mass window must be present there is no analogous parameter to forward or side scatter.Ģ.2. The percentage of each cell type is determined and reported as a percent of the parent cell type.
#FLOWJO 10 FROZEN SOFTWARE#
Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The current mass window selected is approximately AW 78-203, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. We use a Helios CyTOF TM mass cytometer to acquire the ICP-MS data. In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain PBMC that have been previously cryopreserved.