This is the protocol for what we call a checkerboard experiment. It was conceived as a pre-test for high-throughput virus infection screens. In this experiment, cells are seeded at different amounts into a 96-well plate in one direction and incubated with different amounts of virus in the other direction. The purpose is to find the right combination of number of cells and amount of virus, but also to obtain a large single-cell dataset to measure cell-to-cell variability in virus infection and to find possible factors of the population context that determine this cell-to-cell variability.

Note that this experiment is now also routinely performed for different types of cellular activity, either just varying the amount of cells that are seeded followed by a single-cell reporter readout or immunofluorescence staining, or by also varying the amount of a certain stimulus or inhibitor (in analogy to amount of virus), such as a growth factor or a pharmacological compound.

 

Checkerboard protocol “cells/virus” for 96 well plates

This is the layout and protocol of a pre-test for a 96 well plate high-throughput screen. Indicated amounts could be different for different cell types, and should be optimised for each cell type.

CB96

Short protocol:

Day 0:   Seed indicated amount of cells for the indicated celltype on the entire plate.

Day 1: Infect cells with your virus. (Seed less cells for viruses with a long incubation time (24h and more).

Day X: Fix and stain as required. Also fix and stain the “no virus control”. Crucial for correct analysis.

 

Further Checkerboard protocol “siRNA/cells/virus” for 96 well plate

Note: out of the checkerboard “cells/virus” you could perform a further checkerboard to find out the best condition for siRNA-transfected cells and virus transfection. The checkerboard “cells/virus” gives you a first indication of the cell/virus amount to use, in order to reduce the usage of siRNA for this checkerboard. When a transfection is carried out ~20% more cells should be seeded compared to a checkerboard without transfection, in order to obtain a similar number of cells.

This layout is an example, it could obviously vary depending on what you would like to test.

CB96_2

Short protocol:

Day 0: Perform siRNA reverse transfection as described here. Seed indicated amount of cells for the indicated celltype on the wells.

Day 3: Infect cells with your virus. (Seed less cells for viruses with a long incubation time (24h and more).

Day X: Fix and stain as required. Also fix and stain the “no virus control”. Crucial for correct analysis.