Reverse Transfection of Printed microarrays

 

Materials

Reverse Transfection of slides printed with gelatin-DNA

Note: Enhancer, EC buffer and Effectene are components of the Effectene Transfection Reagent kit.

For each slide to be transfected:

  1. In a 1.5 ml micro-centrifuge tube add 16 µl Enhancer to 150 µl EC Buffer.
  2. Mix and incubate for 5 minutes at room temperature.
  3. Add 25 µl Effectene Transfection Reagent and mix by lightly vortexing for 3-4 seconds (setting 4 on Vortex Genie 2, VWR). Steps for both HybriWells and CoverWells are described below. We now routinely use only HybriWells.

The image to the right illustrates how to use HybriWells. Click here for a larger picture.

  1. To use HybriWells (shown in Step 1), peel off the adhesive from the HybriWell and attach the HybriWell over the area of the slide containing the array, making sure an air-tight seal is formed.
  2. Pipet the transfection mix into one of the ports of the HybriWell (Step 2) --the mix will evenly distribute over the array. Depending on the size of HybriWell used volumes may be adjusted appropriately. We use 200µl of transfection mix for the HB2240 HybriWell. Click here for more information on HybriWells.
  3. Expose the array to the transfection reagent for 6-20 minutes (Step 3). For HEK293T cells we find that 15-20 minutes is optimal.
  4. After the incubation with the transfection mix is complete, in a tissue culture hood suction off the mix by inserting into one of the ports the tip of a pasteur pipet attached to a vacuum source (Step 4). Suction off most of the transfection reagent but do not worry about removing the last drop that will remain under the center of the HybriWell.
  5. Pull off the HybriWell using a thin tipped forceps (Step 5). The adhesive attaching it to the slide is strong and a firm and uniform pull is needed to detach the HybriWell.
  6. During the incubation of the printed slide(s) with the transfection reagent, harvest HEK293T cells and suspend 10 x 106 cells in 25 ml medium (DMEM with 10% IFS, 50 units/ml penicillin and 50 µg/ml streptomycin). Click here for how to grow, harvest, and prepare HEK293T cells for reverse transfection. 10 x 106 cells are enough cells for transfecting three slides in one dish.
  7. Place the slide with the printed-side facing up in a sterile 100 x 100 x 10 mm square dish (3 slides fit side-by-side in one plate). In case we are transfecting less than three slides, we use non-printed dummy slides to prevent the printed slides from moving around the dish.
  8. Gently pour the cells onto the slides while avoiding direct pouring on the printed areas (Step 6).
  9. Place the dish in a 37°C, 5% CO2 humidified incubator (Step 7). The microarrays of transfected cells form in ~40 hours and are then ready for use.

The image to the right illustrates how to use CoverWells. Click here for a larger picture.

  1. To use CoverWells, pipet the entire mix from each tube onto a CoverWell (40 x 20 mm) (Step 1).
  2. Gently place a printed slide upside down onto the CoverWell such that the solution covers the entire arrayed area while creating an airtight seal between the slide and the CoverWell (Step 2).
  3. Expose the array to the transfection reagent for 6-20 minutes. For HEK293T cells we find that 15-20 minutes is optimal.
  4. After the incubation with transfection mix is complete, in a tissue culture hood use a thin-tipped forceps to gently pry off the CoverWell from the slide (Step 3). Carefully remove the majority of the remaining transfection mix using a pasteur pipet attached to a vacuum source (Step 4).
  5. Gently pour the cells onto the slides while avoiding direct pouring on the printed areas (Step 5).
  6. Place the dish in a 37°C, 5% CO2 humidified incubator (Step 6). The microarrays of transfected cells form in ~40 hours and are then ready for use.

If required for downstream assays, such as immunofluorescence, fix the slides as follows:

  1. Rinse slides by dipping them for 10 seconds into a Coplin jar containing room temperature PBS.
  2. Transfer the slides into another Coplin jar containing room temperature fixative (PBS with 3.7% paraformaldehyde and 4.0% sucrose)
  3. Fix for 20 minutes.
  4. Before processing by immunofluorescence or other detection methods, rinse the fixed slides for 1-2 minutes in PBS.
  5. Slides can be stored at 4°C in PBS for at least 5 days before processing or examination.
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* Table of Contents *Sabatini Lab *Whitehead Institute for Biomedical Research*