Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

jove.com June 2021 • 172 • e62502 • Page 1 of 15 Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells Paula Ordonez1, Kate N. Bishop2, Jonathan P. Stoye1, Harriet Cordelia Theed Groom3 1 Retrovirus-Host Interactions Laboratory, The Francis Crick Institute 2 Retroviral Replication Laboratory, The Francis Crick Institute 3 Sidney Sussex College, Department of Medicine, University of Cambridge

expressed from an IRES are used to transduce U937 cells. and untransduced cells reveals a restriction ratio. A ratio of 1.0 indicates that the transduced factor has no effect on infectivity. Wild type SAMHD1 expression leads to a fivefold reduction in HIV infection in this assay, corresponding to a restriction ratio of 0.2. While this effect is modest in comparison to more classic restriction factors such as TRIM5, the effect is nonetheless reproducible and allows the classification of modified SAMHD1 expressors into those that restrict in an equivalent manner to the wild-type protein, those that fail to restrict, and those with an intermediate phenotype.
the susceptibility of different tester viruses can be determined.
This protocol outlines the details of virus-like particle (

Protocol
This protocol does not contain any studies involving animals or human participants performed by any of the authors. All the steps of this protocol were performed following the guidelines and codes of practice of the institutions where they were carried out.

Transfection of 293T cells for VLP production (both SAMHD1-expression vectors and tester HIV particles)
NOTE: The most significant contributors to the successful production of virus particles are the health of the producer 293T cells, confluency at transfection and time of harvest.
Laboratories will typically have their preferred transfection reagents and protocol for retroviral particle production. The following protocol yields good quality infectious particles, but equivalent protocols would also be suitable for this application. To maintain good quality 293T cells, passage at least three times per week to maintain a constant growth rate.
Cells should be seeded in the log phase of growth for efficient transfection.
1. Day 1: Seed the required number of 10 cm dishes with a 1/4 split from a near-confluent dish of 293T cells to yield around 60% confluency the following day (approximately 5 x 10 5 cells/mL). Several densities may need to be seeded to achieve an appropriate density for transfection the following day when working with a new cell stock.  2. Add 20 µL of transfection reagent (see Table of Materials), vortex (do not centrifuge) and incubate at 20 °C for 15-20 min. Add the transfection mix dropwise to the plate, swirl gently to mix, and return to the incubator. Alternatives are discussed in the     11. Export the data as .fcs files and clean the fluidics according to local instructions. using appropriate data analysis software.

Representative Results
The rates that are either too high or too low lead to problems with analysis due to lack of uninfected or doubly infected cells.
Equally, a similar transduction rate for SAMHD1 vectors to be compared is key to allowing the same compensation matrix and gating to be applied to the whole experiment, increasing confidence in the subsequent analysis.
Secondly, the age of the U937 cells used is a key factor in whether they differentiate successfully. Successful differentiation can be monitored through observation of adherence, a reduced acidification of the media over time following differentiation and adequate restriction by the wild type positive control in the assay. Differentiation of up to 5 days has been successful.
One of the key advantages to this assay is its flexibility. with various nucleoside reverse transcriptase inhibitors has also been described, and the effect of SAMHD1 shown using this assay modified for use in primary cells 12 , 13 , 14 , 15 .
Adaptation of the protocol for use in primary cells overcomes the limitation posed by examining metabolic phenotypes in transformed cells. However, the existing format allows for a convenient and less technically challenging protocol for high throughput analysis, especially with use of a flow cytometer with a high-throughput sampler. When adapting the protocol, the susceptibility of the internally untransduced cells to bystander effects (e.g., immune signaling) should be considered.
As with many aspects of cell biology, it is essential that such assays are used as part of a holistic approach for understanding a given phenomenon. The parallel use of biochemical assays for SAMHD1 activity 16  Care.