Question: Can I use Flow Sorting with fixed whole blood samples?
Answer: Yes, we developed an immunophenotyping panel that is compatible with our fixation protocols for the Chromium Fixed RNA Profiling (i.e., Single Cell Gene Expression Flex) workflow.
Immunophenotyping Panel
Phenotyping immune populations from the following sample types:
- PBMC (or) Leukocytes Isolated from fixed whole blood (protocol- fixation duration O/N)
- PBMC (or) Leukocytes isolated from fresh whole blood and then fixed (protocol - fixation duration O/N)
Any changes to antibody/fluorophore/clone combinations will have to be validated on either or both sample types, depending on your application.
Cell staining was performed with the following protocol:
- Pellet Approximately 500,000 cells and add 100uL Antibody Mix
- Pipette mix thoroughly
- Incubate @ 4 degree for 30 minutes in the dark
- Stop the reaction with 100uL of 2%FBS+PBS; Spin (400g,5 minutes @4C), discard supernatant
- Wash again with 200uL of 2%FBS+PBS; Spin (400g,5 minutes @4C), discard supernatant
- Resuspend in approximately 200uL volume for up to 500,000 cells appropriate volume and analyze
- We recommend acquiring at least 100,000 total events since some subsets this panel aims to characterize are rare populations
Antibody Mixture
Every antibody in the below panel was titrated individually on the instrument it was intended to be used on. The titrated values are then used to make the antibody mix. Since the final staining volume is 100uL the antibody mix is reconstituted to a final 100uL volume by adding the right amount of cell staining buffer (2%FBS+PBS). In the below panel, the total Antibody volume is 50uL; to make one mixture, add 50uL of staining buffer to achieve a 100uL final volume. It is recommended that this panel be titrated/ validated on the instrument it is intended on being used on.
Please note: We have only validated this panel on the SONY ID7000 spectral Analyzer. Other combinations of the same panel can be used on a conventional cytometer as well but will have to be titrated and validated.
Gating Strategy
Flow Gating Strategy to profile major immune subsets on fresh and fixed samples:
Cell sorting recommendations for processed fixed whole blood
We have demonstrated that processed fixed whole blood can be used to purify PBMCs and Granulocytes using a cell sorter. Once they are moved into the Fixed RNA Profiling Assay, we are able to get comparable PBMC and Granulocyte Biology and complexity metrics when compared to unsorted data.
Instrument
Sort was performed on a MA900 Cell sorter on a 100uM Nozzle; Sort mode was set to purity.
The sample was run at a sample flow rate of approximated 6uL-11uL/minute, which is the lowest sample pressure/flow rate setting (It is recommended to have an optimal cell concentration so that it is possible to sort at a low flow rate and still have an efficient sort time).
Sorted sample was collected in a 1.5mL tube containing PBS + 1% nuclease- free BSA supplemented with RNase inhibitor (Protector RNase inhibitor from Sigma, PN-3335399001). We recommend a final concentration of 0.2 U/ul of RNAse inhibitors.
Samples should be spun down and resuspended in 0.5X PBS + 0.02% nuclease-free BSA before proceeding with probe hybridization.
The sort was done based on differentiating PBMC and Granulocyte subsets based on Forward Scatter (Size) and Side Scatter (Granularity) and purifying based on that.
We have not specifically demonstrated sorting based on epitope markers, but this is low risk and will likely work.
Related Q&A Articles
- Can post Fixation Samples be sorted in the Fixed RNA Profiling Assay
- Flow Cytometry Guidance for Single Cell Protocols
Products: Single Cell Gene Expression Flex