Protocol

Plating Cells for Microinjection

This protocol was adapted from "Gene Delivery by Direct Injection and Facilitation of Expression by Mechanical Stretch," Chapter 4, in Live Cell Imaging (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This protocol describes a method for plating cells for microinjection onto etched coverslips. The coverslips for microinjection must be marked so that microinjected cells can be identified at time points after injection. A procedure for etching coverslips is given below; alternatively, pre-etched coverslips can be purchased at slightly higher cost.

MATERIALS

Reagents

Cell culture for plating

Culture medium

DMEM plus 10% fetal bovine serum (optional; see "Plating Cells," Step 2)

HEPES (pH 7.4) (optional; see "Plating Cells," Step 2)

Equipment

Aluminum foil

Coverslips, number 1, 25-mm square

Diamond pen (e.g., diamond scriber; VWR)

Glass Petri dish, 100 mm

METHOD

Coverslip Etching

If pre-etched coverslips are being used (e.g., CELLocate, Eppendorf), skip below to "Plating Cells."

1. Place coverslips on a solid, clean surface.

2. Use a diamond pen to lightly etch an asymmetric figure onto the coverslip.
If too much pressure is applied while etching, the coverslip will snap in half or will break later in the experiment. However, if too little pressure is applied, the etched figure will be extremely difficult to detect under the microscope, both during microinjection and later when cells are being visualized.

3. Layer etched coverslips onto aluminum wrap that is cut to fit into a 100-mm glass Petri dish. About 10 square coverslips can be placed onto one layer (do not overlap, or they will stick together, rendering them useless). Layer aluminum foil and coverslips to fill the dish, and cover with the glass top (~15 full layers in a dish).

4. Autoclave the coverslips on dry cycle (20 min sterilize, 30 min dry).

Plating Cells

1. Place sterile, etched coverslips into the wells of a six-well plate (one coverslip per well), and plate cells into the wells. Although most cells will adhere to the coverslips, some cells will not (see Troubleshooting).

2. When the cells are at the desired confluency, remove the coverslips from the six-well dish and transfer them to a 60-mm tissue culture dish containing 5 ml of the appropriate medium. Transfer to a microinjection-outfitted microscope for microinjection, as described in Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System and Gene Delivery by Direct Injection (Microinjection) Using a Constant-Flow System.
If injections and time out of the incubator are limited to <20-30 minutes, cells can be maintained without damage in standard medium containing or lacking serum (e.g., DMEM plus 10% fetal bovine serum). However, because the lack of CO₂ will raise the pH of the medium, many investigators perform microinjections in a buffered medium. This is especially important if longer injection times are needed or if the cells being used are especially sensitive to pH. In this case, the medium can be buffered by the addition of 20 mM HEPES (pH 7.4).

TROUBLESHOOTING

Problem: Cells do not adhere well to coverslip.

["Plating Cells," Step 1]

Solution: Two approaches can be taken to promote cell adherence. First, check to see whether the coverslips need to be cleaned; coverslips may have a thin film of grease on them that can limit cell adherence. To clean etched coverslips, incubate them in 2 N NaOH for 2 hours, rinse them extensively with dH₂O, and layer them into a Petri dish for autoclaving (see "Coverslip Etching," Step 3, above). A second way to promote attachment is to coat the coverslips with extracellular matrix proteins, such as MatriGel or rat tail collagen, after autoclaving.