4 Notes
1. The DNA encoding HA-tagged 2x-MCP-2x-GFP was cloned
into the pOPINF vector belonging to the pOPIN vector suite
(https://www.oppf.rc-harwell.ac.uk/OPPF/protocols/clon
ing.jsp) between the NcoI and KpnI restriction sites using
conventional cloning. This plasmid was used as a vector to
generate plasmids expressing the chimeric fusion protein of
POI and 2x-MCP-2x-GFP by inserting the DNA encoding
POI between the KpnI and HindIII sites using ligation-free
cloning. This plasmid as well as the plasmids for expression of
2x-MCP-2x-GFP-RILPL2-RH1 and 2x-MCP-2x-GFP-SKIP
N-terminal domain are available from the Chao lab upon
request.
2. This assay can be performed on a variety of microscope configurations. Here, we provide the details of the microscope used in
the described experiments. Images were acquired on an Olympus IX81 inverted microscope (Olympus) equipped with a
Yokogawa CSU-X1 scanhead (Yokogawa) and Borealis modification (Andor). Our setup consists of a single band-pass filter
or dichroic beam splitter in the scanhead (Semrock Di01T488/568-13x15x0.5). For image acquisition, the emitted
light was passed through an emission filter for GFP (Semrock,
FF01-525/40-25) fluorescence and captured using a backilluminated Evolve Delta EMCCD cameras (Photometrics).
3. Efficient transfection is key to obtain enough cells for imaging.
This protocol is optimized for Lipofectamine 2000, to obtain
efficient transfection without significant cell death. Hence, it is
imperative to optimize for efficient transfection in case a different transfection reagent is used.
4. Seeding of the cells in CYTOO chips is a crucial step for
obtaining enough cells with the desired cellular architecture
for imaging. Seeding too many cells or extremely mild washing
leads to multiple cells attaching to a single pattern eventually
inhibiting the spreading of the cytoplasm of a single cell on the
micropattern. On the other hand, seeding too few cells or
extremely harsh washing leads to detachment of cells from
the micropattern eventually leaving too few cells to image.
ä
Fig. 3 (continued) cell that is transfected with the MCP-GFP-SKIP plasmid is shown. (a and b) For each image,
the arrow indicates the particle being analyzed and the maximum intensity projection of this particle is shown
underneath. The different panels on the right show the output of the HMM-Bayes program including the
schematic of the track being analyzed, the distribution of displacements from the same track, and the
evaluated probability for different modes of movements occurring in this track. The average lifetime for each
type of movement (denoted by τ 1 and τ 2 ) and the respective diffusion coefficients (D, D 1 , and D 2 ) and velocities
in x and y directions (V, V 1 , and V 2 ) are displayed. Note that for purely diffusive particle, the velocity is zero
280
Varun Bhaskar et al.
1. The DNA encoding HA-tagged 2x-MCP-2x-GFP was cloned
into the pOPINF vector belonging to the pOPIN vector suite
(https://www.oppf.rc-harwell.ac.uk/OPPF/protocols/clon
ing.jsp) between the NcoI and KpnI restriction sites using
conventional cloning. This plasmid was used as a vector to
generate plasmids expressing the chimeric fusion protein of
POI and 2x-MCP-2x-GFP by inserting the DNA encoding
POI between the KpnI and HindIII sites using ligation-free
cloning. This plasmid as well as the plasmids for expression of
2x-MCP-2x-GFP-RILPL2-RH1 and 2x-MCP-2x-GFP-SKIP
N-terminal domain are available from the Chao lab upon
request.
2. This assay can be performed on a variety of microscope configurations. Here, we provide the details of the microscope used in
the described experiments. Images were acquired on an Olympus IX81 inverted microscope (Olympus) equipped with a
Yokogawa CSU-X1 scanhead (Yokogawa) and Borealis modification (Andor). Our setup consists of a single band-pass filter
or dichroic beam splitter in the scanhead (Semrock Di01T488/568-13x15x0.5). For image acquisition, the emitted
light was passed through an emission filter for GFP (Semrock,
FF01-525/40-25) fluorescence and captured using a backilluminated Evolve Delta EMCCD cameras (Photometrics).
3. Efficient transfection is key to obtain enough cells for imaging.
This protocol is optimized for Lipofectamine 2000, to obtain
efficient transfection without significant cell death. Hence, it is
imperative to optimize for efficient transfection in case a different transfection reagent is used.
4. Seeding of the cells in CYTOO chips is a crucial step for
obtaining enough cells with the desired cellular architecture
for imaging. Seeding too many cells or extremely mild washing
leads to multiple cells attaching to a single pattern eventually
inhibiting the spreading of the cytoplasm of a single cell on the
micropattern. On the other hand, seeding too few cells or
extremely harsh washing leads to detachment of cells from
the micropattern eventually leaving too few cells to image.
ä
Fig. 3 (continued) cell that is transfected with the MCP-GFP-SKIP plasmid is shown. (a and b) For each image,
the arrow indicates the particle being analyzed and the maximum intensity projection of this particle is shown
underneath. The different panels on the right show the output of the HMM-Bayes program including the
schematic of the track being analyzed, the distribution of displacements from the same track, and the
evaluated probability for different modes of movements occurring in this track. The average lifetime for each
type of movement (denoted by τ 1 and τ 2 ) and the respective diffusion coefficients (D, D 1 , and D 2 ) and velocities
in x and y directions (V, V 1 , and V 2 ) are displayed. Note that for purely diffusive particle, the velocity is zero
280
Varun Bhaskar et al.