Prepared by: Christine Muñoz
Protocol for Culturing Electrocytes
-Surgical tool kit (forceps, Vannas)
-Supplemented Dubelcco’s Modified Eagle’s Medium (DMEM)
-Modified Sterno Saline
-1 35 mm Petri dish with Sylgard and pins
-1 35 mm Petri dish with coverglass (coated with PDL or not)
-Stocked Laminar Flow hood
IMPORTANT: Follow aseptic procedure protocol in every part of this procedure; when culturing cells an aseptic environment is of utmost importance.
-Before beginning any type of work concerning tissue culture wipe hands down with 70% ethanol put gloves on and wipe gloves down with 95% ethanol. Any time hands are removed from under the hood, or when contamination is suspected, wipe gloves down with 95% ethanol before placing them back under the hood.
-Wipe down work surface with ethanol before and after work.
-Before placing anything under the hood, such as bottles or surgical instrument, wipe down with ethanol. If this proves to be an inefficient way of sterilizing equipment, items to be used can be placed under the hood and UV irradiated for 25-30 min. before estimated time of use.
-Flame bottle caps when opening and closing sterile bottles or conical tubes (makes sure the ethanol has evaporated). When keeping a bottle open place the bottle cap upside down on a surface that has been recently wiped down with ethanol.
-Do not pour liquids out of the mouth of the bottle. Use a pipette to remove liquid from a bottle and avoid touching the pipette to the mouth of the bottle. If in doubt throw the pipette away and use a new one.
-When distributing liquid from one bottle amongst many receptacles flame the tip of the pipette before drawing more liquid out of the original bottle.
-Avoid passing over containers when possible, instead move around them. This is especially important when Petri dishes containing cells are open.
Preparation of Supplemented Media (and Glucose Supplement)
-Using a pipette remove 75 mL of media from new DMEM bottle and discard. -Add the following to the media bottle:
; 5 mL L-glutamine
; 10 mL Glucose (30%)
; 5 mL Non-essential amino acids
; 50 mL Fetal bovine serum
; 5 mL Antibiotic/antimycotic
To prepare 30% Glucose:
Add 75mg of D-glucose to 250 mL of dHO (autoclaved), stir until dissolved. 2
Filter sterilize the mixture and aliquot into 10 mL volumes. This volume of 30% glucose added to the bottle of DMEM will yield a 32 mM solution at final volume.
Preparing Lynne McAnelly’s Simple Sterno Saline
Product mM mOsm Formula Wt. g/L NaCl 114 228 58.44 6.66
KCl 2 4 74.50 0.15
CaCl•HO 4 12 147.00 0.59 22
MgCl• HO 2 6 203.30 0.41 22
HEPES 5 10 238.30 1.19
Titrate to pH 7.2 with NaOH (6M) then Autoclave
You can later add:
Glucose 5 5 180.20 0.90
Petri Dishes with Coverglass (Both Coated and Uncoated)
-Dip coverglass in 200 proof ethanol, dab off excess alcohol and flame. -Place coverglass in Petri dish.
-UV irradiate sterilized coverglass in dish (25-30 min.).
**Stop here for uncoated coverglass, continue for Poly-D-Lysine for attachment matrix coating on coverglass**
-Coat coverglass with enough working solution of Poly-d-lysine to coat entire coverglass. -Incubate at room temperature for 5-15 minutes.
-Aspirate Poly-d-lysine with vacuum system, or take up in pipette and reuse on other coverglass.
-Rinse with sterile water (two or three times).
-Plates can be used immediately or store under sterile condition (wrapped in aluminum foil).
PDL stock solution
-Mix 10mg/10mL dHO (autoclaved) until completely dissolved. 2
-Store at -20?C in appropriate aliquots.
PDL working solution (0.1 mg/mL)
-For every 1mL of PDL stock solution add 10mL dHO (autoclaved). 2
Proper Setup under the Laminar Flow Hood (See figure 1)
2) Autoclaved Pasteur pipettes
3) 95% alcohol
5) Alcohol lamp
6) Vacuum setup (Erlenmeyer flask, plastic tubing, curved glass attachments, stopper with two holes, pipette attachment)
8) Permanent Marker
Items to have close at hand: Individually wrapped pipettes (5/10/25 mL)
Obtaining Electrocytes from Pinni
-With Vannas scissors quickly cut off a 1-1.5 cm potion from the tip of the tail. -Place the tail section in the Petri dish lined with Sylgard containing 5-6 mL of modified Sterno Saline.
***Continue work under Laminar Flow Hood- Fan on, UV light off
-Anchor top left section of the tail with a minutiae pin anchor the bottom right with another minutiae pin. (Fig 2a)
-With Vannas scissor, and forceps to hold the tissue in place, cut the skin on the side of the tail section horizontally. (Fig 2b)
-Flip the tail section 180? clockwise and pin down as before. (Fig 2c)
-Again with the Vannas scissors and forceps in hand cut the skin on the other side of the tail section horizontally. With the forceps pull the loose skin off the tail section to expose the electrocytes. (Fig 2d)
-Place recently exposed electrocytes in a Petri dish containing 5-6 mL of supplemented DMEM (warmed to room temperature).
-Keep the Petri dish under the laminar flow hood at room temperature with the fan on and UV light off.
Change of Media for Electrocytes in Culture
-Using the vacuum setup found under the hood and autoclaved Pasteur pipettes (flame the tip of the pipette with alcohol lamp) aspirate the media out of the Petri dish containing electrocytes.
-Replace the media with 5-6 mL of newly supplemented media warmed to room temperature.
-Replace the media every other day, or sooner if necessary (media turns a straw color if nutrients are depleted).
Dissociation of Electrocytes
-Place tail section in a 35 mm Petri dish containing 5-6 mL of modified Sterno saline containing 1% (w/v) crude collagenase for 4-7 days at 6?C or at room temperature for a
few hours (must be tested).
-Gently triturate saline to help with the dispersal of the collagenase-treated electrocytes.