While primary culture of cells derived from the central nervous system is a powerful tool, the resulting lack of cellular heterogeneity or three-dimensional structure can render these approaches limited. One way to address these shortcomings is through the use of organotypic brain slice cultures, which preserve neural architecture and diversity for downstream analyses. Transfection of these cultures, however, can be both challenging and frustrating, which is why Dr. Maik Hintze (University of Bonn) developed this transfection protocol using the Xfect Transfection Reagent:

Cerebellar slice cultures are not very easy to transfect, often making viral transduction necessary. We have tested several transfection protocols that would give us good transfection efficiencies while allowing single-cell separation for counting and morphology. The Xfect Protocol provides a perfect balance of high cell numbers and good single-cell separation which perfectly matches our needs. The protocol is fast and simple to perform."

—Dr. Maik Hintze (University of Bonn)

A. Prior to transfection

1. Prepare Culture Medium as follows:
   
   

   MEM, HEPES, no glutamine (Thermo Fisher Scientific, Cat. # 32360034)	100 ml
   L-Glutamine (200 mM) (Thermo Fisher Scientific, Cat. # 25030081)	1 ml
   HBSS, calcium, magnesium (Thermo Fisher Scientific, Cat. # 24020133)	50 ml
   Horse Serum, heat inactivated, New Zealand origin (Thermo Fisher Scientific, Cat. # 26050088)	50 ml
   Penicillin-streptomycin (10,000 units of each in 0.85% NaCl)	2 ml
   Total volume	203 ml

2. Less than 4 hr prior to starting the transfection, transfer freshly prepared organotypic cerebellar slice cultures onto Millipore culture membranes (Biopore Membrane Filter roll, Hydrophilic PTFE; Millipore Sigma, Cat. # BGCM00010) and place on top of a Millipore culture dish insert (Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE; Millipore Sigma, Cat. # PICMORG50). Allow slices to equilibrate in OptiMEM for at least 2 hours.
   
   

   NOTE: This dual-membrane approach improves transfection efficiency by slowing the rate at which the transfection reagent drains from the tissue into the underlying medium.

B. Prepare transfection mixture

Prior to beginning your transfection, consult the table below to determine the total required reagent volumes.

1. Add DNA to a sterile 1.5-ml microcentrifuge tube and bring up to the indicated volume with Xfect Reaction Buffer. Mix by pipetting.
2. Add the indicated volume of Xfect Polymer and mix well by vortexing for 5 sec.
3. Incubate the mixture for 10 min at room temperature.
4. During this incubation period, aliquot the indicated volume of OptiMEM (Thermo Fisher Scientific, Cat. # 31985062) into a separate, sterile, 1.5-ml microcentrifuge tube.
5. After the 10-min incubation has passed, add the mixture from Step 3 to the OptiMEM and mix well by vortexing for 5 sec.

C. Transfect organotypic slice cultures

1. Pipette 15 µl of the transfection mixture (Section B, Step 5) directly onto each prepared organotypic cerebellar slice (Section A, Step 2).
2. While cerebellar slices are undergoing transfection, equilibrate Culture Medium for at least 2 hr.
   
   

   NOTE: As an optional step, you may wash each slice with 50–100 µl of OptiMEM 4 hr after transfection. Aspirate after 1 min. Use minimal OptiMEM in this step to avoid causing slices to float off the membrane/culture dish insert. Proceed immediately to Step 3.

3. 4 hr after transfection, transfer membranes with organotypic slices from OptiMEM to a well containing the equilibrated Culture Medium.