Obtaining a Single-Cell Suspension from Frozen Rat Brain Tissue

Abstract

Source: Rubio, F. J., et. al. Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue. J. Vis. Exp. (2016)

This video demonstrates the dissociation of frozen rat brain tissue into a single-cell suspension using enzymatic digestion and mechanical trituration. The prepared cells are fixed and permeabilized with ethanol for downstream applications like flow cytometry or gene expression analysis.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review.

1. Preparation Before Tissue Collection

1. Set the centrifuge to 4 ^oC.
2. Fire polish a set of three glass Pasteur pipettes with decreasing diameters of approximately 1.3, 0.8, and 0.4 mm for each sample.
3. Prepare labeled 1.7 ml tubes containing 1 ml cold Buffer A and keep the tubes on ice.
4. Prepare an ice tray containing the brain slicing matrix, spatulas and glass plates (or inverted glass Petri dish) on which to perform the dissection. Pre-chill two or more razor blades on the glass plates and use tissues paper to dry all instruments that touch the tissue.
5. Thaw the Enzyme solution at room temperature (RT) for 30 min before tissue collection.

2. Tissue Collection and Dissection

1. Initiate the behavioral or pharmacological treatment 90 min before tissue collection.
   NOTE: For the results shown here, acute intraperitoneal injections of 5 mg/kg methamphetamine on rats in a novel environment (test condition) and naïve rats kept in their home cages (control condition) were performed.
2. Anesthetize the rat by placing it in a glass desiccator jar with saturated isoflurane and decapitate the rat 30 - 60 sec later using a guillotine.
   Use scissors to remove the skin and muscle from the head and expose the skull. Use rongeurs to cut the skull, open up the foramen magnum, and remove the back part of the skull. Use rongeurs to cut along the top edges of the skull to expose the brain. Be careful not to damage the brain. Use a small spatula to gently scoop under and elevate the brain. Raise the brain and cut the nerves until the brain is free.
3. Dissect tissue using razor blades.
   NOTE: Obtain tissue samples using either of the following methods: (2.3.1) freshly dissected tissue; (2.3.2) frozen tissue after dissection; or (2.3.3) frozen tissue dissected from frozen whole brain. Keep the brain slicing matrix, razor blades and glass plate dry throughout the dissection and mincing processes as condensed water can lead to hypotonic lysing of cells. Use magnifying glasses to ensure accurate dissection.
   1. For freshly dissected tissue, place the freshly extracted brain into a brain-slicing matrix (a rat brain-shaped metal mold with slots for the razor blades at 1 mm intervals) that has been chilled on ice. Insert two or more pre-chilled razor blades into the slots to cut coronal slices that contain the brain region(s) of interest. Place the cut slice on the chilled glass plate.
      Note: Dissect the brain regions of interest on a chilled glass plate.
   2. For frozen tissue after dissection, dissect the brain region(s) of interest from slices of freshly extracted brain, similar to that described above in 2.3.1. Place the dissected tissue into a microtube and rapidly freeze the tissue by submerging the microtube in -40 ºC isopentane for 20 sec. Keep dissected tissue frozen at all times in a -80 ºC freezer until further processing.
   3. For frozen tissue, immediately freeze the freshly extracted brain in -40 ºC isopentane and store in a sealed bag at -80 ºC for up to 6 months.
      1. On the day of dissection, place the frozen brain in a cryostat set at approximately -20 ºC (no warmer than -18 ºC) for approximately 2 hr to equilibrate the temperature.
         NOTE: Brains can be cut easily at this temperature, while much cooler temperatures make the brain too brittle to be cut safely.
      2. Use razor blades to cut 1 - 2 mm coronal slices of the frozen brains in a cryostat. Under freezing conditions, use a blunted 12—to 16-gauge needle to obtain tissue punches from these slices. Keep dissected tissue frozen at all times until further processing.
   4. Add 1 - 2 drops of Buffer A to cover the dissected tissue before mincing. Remove most of the white matter from the tissue using two razor blades to prevent the loss of neurons during the rest of the trituration process. If starting with frozen tissue, then allow the tissue to thaw on the cold glass plate for no more than 1 min before applying Buffer A solution.
   5. Mince the tissue 100 times in each orthogonal direction with a razor blade on a chilled glass plate. Hold the razor blade vertically to the glass plate when mincing.
      NOTE: Thorough mincing is critical for all protocol steps.
   6. Use razor blades to transfer the minced tissue into microcentrifuge tubes containing 1 ml of cold Buffer A. Invert the tube 3 - 5 times to keep all minced tissue in the solution.

3. Cell Dissociation

NOTE: Use gentle end over end mixing for all of the following steps. Do not use a vortex mixer and avoid air bubbles that cause cell damage.

1. Centrifuge the sample tubes at 110 x g for 2 min at 4 ºC. Discard supernatant. Slowly add 1 ml of cold, freshly thawed Enzyme solution (containing a mix of proteolytic enzymes) down the inner wall of the microtube. Immediately suck up the entire pellet and gently pipette up and down only 4 times with a moderately large tip diameter pipette to disperse the minced tissue pellet.
2. Invert the microtubes immediately to prevent the pellet from sticking to the bottom, and incubate the samples with end-over-end mixing for 30 min at 4 ^oC.
3. After enzymatic tissue digestion, centrifuge the tubes at 960 x g for 2 min at 4 ºC. Remove the supernatant and add 0.6 ml of cold Buffer A. Immediately after adding Buffer A, use the same pipette tip to disperse the pellet by sucking up the entire pellet and gently pipette up and down 5 times.
4. Mechanically triturate the digested tissue and collect in 15 ml tubes using the following steps. For each sample, use a separate set of 3 fire-polished glass Pasteur pipettes with descending diameters of approximately 1.3, 0.8, and 0.4 mm attached to latex bulbs.
   1. Gently triturate each sample 10 times with the 1.3 mm glass pipette. Let the sample settle for 2 min on ice (the debris and undissociated cells will settle to the bottom). Collect the supernatant (~ 0.6 ml) and transfer it to a 15 ml conical tube (tube #1).
   2. Add 0.6 ml Buffer A solution to the remaining pellet. Triturate the sample 10 times with the 0.8 mm glass pipette. Let the sample settle for 2 min on ice. Collect the supernatant (~ 0.6 ml) and transfer it to the same 15 ml conical tube #1.
   3. Add 0.6 ml Buffer A solution to the remaining pellet. Triturate the sample 10 times with the 0.4 mm glass pipette. Let the sample settle for 2 min on ice. Collect the supernatant (~ 0.6 ml; without touching the pellet with non-dissociated cells) and transfer to the same 15 ml conical tube #1.
      NOTE: Keep the 0.4 mm diameter pipette in the tube #1 for the following trituration steps.
   4. Repeat step 3.4.3 three more times using the smallest glass pipette (~ 0.4 mm diameter), and collect the supernatant into a separate 15 ml conical tube (tube #2).
   5. Triturate the collected cell suspensions in tubes #1 and #2 for 10 more times using the 0.4 mm glass pipette and keep them on ice.

4. Cell Fixation and Permeabilization

1. Prepare 4 microtubes per sample (two microtubes for cells from tube #1 and two for cells from tube #2) by adding 800 µl of 100% cold ethanol (stored at -20 ^oC before use) into each tube and keep them on ice. Note: The final ethanol concentration will be 50%.
2. Pipette ~ 800 µl of the cell suspensions (from tube#1 and tube#2) into each of the 4 tubes containing cold ethanol and invert the tubes to mix the samples.
3. Incubate tubes on ice for 15 min while inverting the tubes every 5 min.
4. After fixation/permeabilization, centrifuge the tubes at 1,700 x g for 4 min at 4 ºC and discard the supernatant. Leave 50 µl of solution to prevent cell loss.
   NOTE: The pellets at this stage are white and stick less to the wall of the tubes than before permeabilization. Therefore, remove the supernatant carefully by touching the wall of the microtube where there is no pellet and draw up the supernatant slowly using a micropipette.

5. Cell Filtration

1. Re-suspend the pellets from step 4.4 with cold PBS as follows:
   1. Add 550 μl of cold PBS to one of the two microtubes coming from tube #1, and use a moderately large diameter pipette tip to gently pipette the cell suspension up and down 5 times. Repeat the same for one microtube coming from tube #2.
   2. For cells from tube #1, using the same pipette, transfer the first cell suspension to the second pellet. Resuspend the second pellet by gently pipetting the combined cell suspension up and down 5 times.
   3. For cells from tube #2, resuspend and combine the pellets (i.e., third and fourth microtubes) as described in 5.1.2.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Brain matrix	CellPoint Scientific	69-2160-1	To obtain coronal brain slices
Hibernate A low fluorescence	Brain Bits	HA-lf	Buffer A' in the protocol is used for processing tissue and cells from the dissociation to fixation steps
Accutase	Millipore	SCR005	Enzyme solution' in the protocol is used for enzymatic digestion of tissue prior to trituration
Cell Strainer, 40 µm	BD Falcon	352340	To filter cell suspension
Cell Strainer, 100 µm	BD Falcon	352360	To filter cell suspension
Falcon 5 ml round-bottom polystyrene test tube with cell strainer snap cap	BD Bioscience	352235	To filter cell suspension before passing though the flow cytometer
Pasteur Pipet, Glass	NIH supply	6640-00-782-6008	To do tissue trituration