TITLE: Isolation of sex specific PND 1/2 rat cortical primary astrocytes
AUTHORS: Neha Paranjape, Jonathan A. Doorn, Hansjoachim Lehmler, Laura E Dean

[DESCRIPTION]
Many xenobiotic metabolizing enzymes, such as P450s, SULTs, UGTs, and sulfatases, are expressed in the brain, including astrocytes, the most abundant cell type in the brain. To obtain a purified culture of primary astrocytes, a method was developed and confirmed through flow cytometry. Primary glial cells are isolated separately from male and female postnatal day 1/2 (PND1/2) rat pups to isolate developing astrocytes. Sex is first determined by the anogenital distance and then confirmed through PCR followed by gel electrophoresis. Once isolated, the medium is changed every 3-4 days with additional incubation at room temperature for 10 min on a plate shaker before aspirating media to release microglia. When flasks reach confluency, cells are passaged and reseeded with 2 x 105 cells per flask. Once the cells reached passage 3, or appear to be confluent with only astrocytes, flow cytometry is performed utilizing Anti-Iba1 and GFAP primary antibodies.

[STEPS]
SECTION: Before you begin
1. Prepare complete S-MEM
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1.1. Add 5 mL pen/strep solution to the 500 mL S-MEM.
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1.2. Store complete S-MEM medium for up to 2 months at 4ºC 
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2. Prepare dissociation medium and warm to 37ºC - MUST MAKE FRESH EACH ISOLATION DAY. Prepare 10 mL of dissociation medium per sex per litter of PND1/2 pups (20 mL total).
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2.1. Add 187.5 U dispase for cortex isolation per 125 mL complete S-MEM in an autoclaved container.
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2.2. Sterilize with a 150 mL sterile filter flask (0.2 μm pore size, cellulose acetate)
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2.3. Prewarm in 37ºC hot water bath for 1 hour before use.
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3. Prepare astrocyte growth medium.
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3.1. Add 900 mL MEM, 10 mL pen/strep, and 100 mL heat-inactivated horse serum to a 1 L sterile filter flask.
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3.2. Sterilize with a 1 L sterile filter flask (0.2 μm pore size, cellulose acetate) by pulling vacuum through the apparatus.
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3.3. Store the astrocyte growth medium for up to 1 month at 4ºC.
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4. Prepare DNase I solution, 8000 U/mL.
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4.1. Add 15,000 U DNase I from bovine pancreas (type IV) to 1.875 mL autoclaved water.
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4.2. Mix gently, do NOT vortex. DNase I from pancreas is vulnerable to inactivation by physical damage and should not be vortexed.
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4.3. Sterilize with a 0.2 μm syringe filter (cellulose acetate).
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4.4. Store in 100 μL aliquots at -20ºC.
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5. Fire polish and Sigmacote treatment of pipets for cell isolation.
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5.1. Fire polish the tip of a 9-in Pasteur pipet by placing the small end in an open flame from a Bunsen burner for a few seconds. This will slightly melt the glass and will produce a smooth tip to the pipet, which helps prevent damage to the cells. However, if the opening becomes too small, increased shear forces during titration may lead to increased cell damage.
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5.2. Plug the large end of the fire-polished Pasteur pipets with cotton removed from a cotton-tipped swab or, alternatively, small pieces of cotton balls.
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5.3. Treat fire-polished Pasteur pipets and 10 mL glass, cotton-plugged serological pipets with Sigmacote by drawing the viscous Sigmacote solution up into the pipet with a portable power pipettor. Do not let the solution touch the cotton plug. Allow Sigmacote to drain back into the bottle, and allow the pipets to drain and dry completely in a beaker lined with paper towels.
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5.4. Place Sigmacoted pipets in a metal container with lid and autoclave.
SECTION: Brain and Tail Dissection
7. Sex the pups based on anogenital distance (females will have a much shorter distance than males, compare pups to one another to be able to sex pups efficiently).
SECTION: Before you begin
6. Autoclave fine-angled micro-dissecting scissors, large scissors, and forceps in a beaker wrapped with aluminum foil.
SECTION: Brain and Tail Dissection
9. Place the head on a sterile gauze pad. Each head will be placed on a separate gauze pad.
SECTION: Brain and Tail Dissection
11. Remove brain from head.
SECTION: Brain and Tail Dissection
11.1. Secure the head by holding down the snout.
SECTION: Brain and Tail Dissection
11.2. With fine-angled micro-dissecting scissors, cut the skin along the midline from the base of the skull to the eyes.
SECTION: Brain and Tail Dissection
11.3. Use 0.8 mm forceps to separate skin and expose the skull as necessary.
SECTION: Brain and Tail Dissection
11.4. Cut skull along midline with micro-dissecting scissors.
SECTION: Brain and Tail Dissection
11.5. Cut away the skull flaps for easier removal of the brain, remove the brain with spatula, and place the brain on a sterile gauze pad. 
SECTION: Brain Tissue Dissection for Cortex
12. Split the brain in half down the midline to separate each hemisphere from one another. Place each hemisphere with the inside facing up to examine the hippocampus on the sterile gauze pad.
SECTION: Brain Tissue Dissection for Cortex
13. Gently remove darker hippocampal crescents with forceps (dissecting microscope can be used here to assist in locating the different brain regions).
SECTION: Brain Tissue Dissection for Cortex
14. Remove remaining meninges with forceps (look for any veins and flip the brain section over a few times to use the sterile gauze pad to help remove the meninges).
SECTION: Brain Tissue Dissection for Cortex
15. Place sections in a sterile 50 mL conical polypropylene tube containing 10 mL complete S-MEM on ice labeled with the proper sex
SECTION: Brain and Tail Dissection
8. For pups at PND1/2, use between 8 and 15 pups and decapitate using sterile large scissors.
SECTION: Brain Tissue Dissection for Cortex
16. Repeat steps, placing all cortical sections in a single 50 mL tube on ice per sex (all pups from the same litter with the same sex can be placed in the same 50 mL tube).
SECTION: Brain and Tail Dissection
10. Take a tail snip of approximately 0.5-1 cm (too much tissue may not be digested with the DNA extraction kit in the steps to follow) and place the tail in a 1.5 mL centrifuge tube, label the tube according to the sex of the pup, and then place it on ice.
SECTION: Confirm sex of pups
17. Add 100 μL extraction solution from Extract-N-Amp Tissue PCR kit to each centrifuge tube containing a small tail snip for a pup (collected in step 10).
SECTION: Confirm sex of pups
18. Add 25 μL tissue preparation solution from Extract-N-Amp Tissue PCR kit
SECTION: Confirm sex of pups
19. Incubate at room temperature for 10 min
SECTION: Confirm sex of pups
20. Incubate in 95ºC hot water bath for 3 min
SECTION: Confirm sex of pups
21. Remove from hot water bath and add 100 μL neutralization solution B from Extract-N-Amp Tissue PCR kit
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22. Vortex
SECTION: Confirm sex of pups
24. Store tissue extract at 4ºC or use for PCR immediately
SECTION: Confirm sex of pups
25. PCR amplification of Sry gene
SECTION: Confirm sex of pups
25.1. Prepare master mix with the following per tissue extract:  10 μL extract-N-amp PCR reaction mix, 4 μL RNase- and DNase-free water, 1 μL of 25 μM forward primer (5’- TAC AGC CTG AGG ACA TAT TA -3’), 1 μL of 25 μL reverse primer (5’- GCA CTT TAA CCC TTC GAT GA -3'). An autosomal gene, Actb that encodes for beta-actin was used as control with the following set of primers:  Forward: 5’- AGC CAT GTA CGT AGC CAT CC -3’; Reverse: 5’- TGT GGT GGT GAA GCT GTA GC -3’.
SECTION: Confirm sex of pups
25.2. To PCR tubes labeled for each tail snip extract, add 16 μL of master mix 
SECTION: Confirm sex of pups
23. Remove tail snip and discard
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25.3. Add 4 μL tissue extract to each designated PCR tube containing the master mix
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26. Run PCR program
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26.1. 94ºC for 5 min
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26.2. 35 cycles of:  94ºC for 30 seconds, 50ºC for 30 seconds, and 72ºC for 1 min
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26.3. 72ºC for 7 min
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27. Prepare 1% agarose gel
SECTION: Confirm sex of pups
27.1. Prepare 1x TAE buffer by diluting 10x TAE buffer with DI water (1:10).
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27.2. Add 1 g agarose to 100 mL 1x TAE buffer
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27.3. Microwave in 1 min intervals, swirling the flask each min until clear
SECTION: Confirm sex of pups
27.4. Let agarose cool to ~50ºC (warm to touch and able to grab)
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27.5. Add 2 μL EtBR or use another nucleic acid stain, such as GelRed per manufacturer's instructions such that final stain concentration is 1x.
SECTION: Confirm sex of pups
27.6. Pour gel into gel tray with well comb(s) in place
SECTION: Confirm sex of pups
27.7. Let cool at room temperature for 1 hour (until solidified)
SECTION: Confirm sex of pups
28. Fill the gel electrophoresis box with 1xTAE buffer until the gel is fully covered
SECTION: Confirm sex of pups
29. To each PCR-amplified DNA sample (20 μL), add 4 μL of loading dye (6x concentration).
SECTION: Confirm sex of pups
30. Carefully load 10 μL of the ladder followed by samples to each well
SECTION: Confirm sex of pups
31. Run gel at 100 V for 1 hour
SECTION: Confirm sex of pups
32. Turn the power off and disconnect the electrodes
SECTION: Confirm sex of pups
33. Visualize under UV light to confirm sex of pups. The samples for males should show a band for Sry gene, amplified product of 317 bp while all samples should show a band for Actb gene, amplified product of 220 bp.
 

SECTION: Dissociate cells extraction
34. Carefully remove as much complete S-MEM as possible with a sterile, cotton-plugged, 9' Pasteur pipet, taking care to retain all pooled brain sections.
SECTION: Dissociate cells extraction
35. Add 7 mL prewarmed dissociation medium to a 50 mL sterile beaker with a stir bar and carefully pour sections into the beaker.
SECTION: Dissociate cells extraction
36. Gently triturate the sections 7-8 times using a Sigmacote-treated 10 mL serological pipet.
SECTION: Dissociate cells extraction
37. Stir for 10 min at low speed on a stir plate inside the biosafety cabinet.
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38. While this is stirring, prepare 5 of the 15 mL centrifuge tubes for each extraction by adding 5 mL room temperature astrocyte growth medium to each tube.
SECTION: Dissociate cells extraction
39. Additionally, thaw DNase I solution and place on ice
SECTION: Dissociate cells extraction
40. After 10 min of gentle stirring, remove the beaker from the stir plate and place at ~45º angle for 3 min to allow the non-dissociated tissue to collect at the bottom of the beaker (e.g. using a 1.5 mL centrifuge tube holder).
SECTION: Dissociate cells extraction
41. Carefully aspirate the dissociated cells in medium with a Sigmacote-treated 10 mL glass serological pipet (take care not to remove the undissociated tissue pieces).
SECTION: Dissociate cells extraction
42. Place this suspension into a 15 mL centrifuge tube that contains astrocyte growth medium
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43. Invert the 15 mL centrifuge tube 2-3 times to mix and then allow the tube to sit undisturbed during the continuing extractions.
SECTION: Dissociate cells extraction
44. Add 5 mL prewarmed dissociation medium to the 50 mL beaker containing undissociated tissue pieces.
SECTION: Dissociate cells extraction
45. Add 100 μL of 8000 U/mL DNase I solution
SECTION: Dissociate cells extraction
46. Stir for 10 min at low speed on a stir plate inside the biosafety cabinet.
SECTION: Dissociate cells extraction
47. After 10 min of gentle stirring, remove the beaker from the stir plate and place at ~45º angle for 3 min to allow the non-dissociated tissue to collect at the bottom of the beaker (using a 1.5 mL centrifuge tube holder works nicely here).
SECTION: Dissociate cells extraction
48. Carefully aspirate the dissociated cells in medium with a Sigmacote-treated 10 mL glass serological pipette (take care not to remove the undissociated tissue pieces).
SECTION: Dissociate cells extraction
49. Place this suspension into a 15 mL centrifuge tube that contains astrocyte growth medium.
SECTION: Dissociate cells extraction
50. Invert the 15 mL centrifuge tube 2-3 times to mix and then allow the tube to sit undisturbed during the continuing extractions.
SECTION: Dissociate cells extraction
51. Add 5 mL prewarmed dissociation medium to the 50 mL beaker containing undissociated tissue pieces. Do not add any more DNase I solution for the remaining extractions.
SECTION: Dissociate cells extraction
52. Once you have dissociated a total of 3 times, remove undissociated tissue from the 15 mL tubes by inserting a sterile, fire-polished, cotton-plugged, Sigmacote-treated 9" Pasteur pipette to the bottom of the tube and carefully aspirate the undissociated tissue. 
SECTION: Dissociate cells extraction
53. Place this tissue back into the 50 mL beaker and perform the final two extractions, adding another 5 mL prewarmed dissociation medium to the 50 mL beaker each time. 
SECTION: Pellet Cells
54. Pool dissociated cells and medium from all 15 mL centrifuge tubes into a singular 50 mL conical tube.
SECTION: Pellet Cells
55. Centrifuge for 10 min at 1000 rcf at 4ºC, in a swinging bucket rotor to pellet the suspended cells.
SECTION: Pellet Cells
56. Carefully aspirate medium from the cell pellet.
SECTION: Pellet Cells
57. Resuspend in 20 mL astrocyte growth medium by gently pipetting with a Sigmacote-treated 10 mL glass serological pipette.
SECTION: Pellet Cells
58. Allow cells to sit for 5 min and then discard any sedimented tissue from the bottom of the tube by using a fire-polished Sigmacoted 9" Pasteur pipette.
SECTION: Assess cell number and viability
59. Gently mix 100 μL cell suspension with 100 μL of 0.08% trypan blue staining solution
SECTION: Assess cell number and viability
60. Allow cells to take up the trypan blue for 3 min
SECTION: Assess cell number and viability
61. Determine the total cell number and cell viability with a hemacytometer and an inverted phase-contrast microscope (intact cells are able to exclude trypan blue while dead or damaged cells retain the dye).
SECTION: Plate and grow cells
62. Plate 5+ T75 cell culture flasks with 7.5x106 cells per flask in 9 mL astrocyte growth medium.
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63. Place plated cells in a 37°C, 95% air/5% CO2, 95% relative humidity incubator.
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64. After 18-24 hours, remove the old growth medium with a sterile 5" Pasteur pipette attached to a vacuum source.
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65. Add 9 mL of fresh astrocyte growth medium to each flask.
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66. Change medium twice per week (every 3-4 days) as described below.
SECTION: Plate and grow cells
66.1. For primary astrocytes that are passage 0-2 (P0-P2), check cells under the microscope, shake flasks for 10 minutes at a medium-low speed on a plate shaker, check cells under the microscope again to make sure astrocytes are still adhering to the flask while the microglia have popped off, aspirate media, add 5 mL DPBS per T75 flask, rinse plate well by moving DPBS back and forth across flask bottom, aspirate, add 9 mL growth media per T75 flask, and then place back in the 37°C, 95% air/5% CO2, 95% relative humidity incubator.
  

SECTION: Plate and grow cells
66.2. For primary astrocytes that are either confluent astrocytes, or are passage 3+ (P3+), check cells under the microscope, aspirate, add 9 mL growth media per T75 flask, check cells once again under the microscope, and then place back in the 37°C, 95% air/5% CO2, 95% relative humidity incubator.
  

SECTION: Flow Cytometry to confirm purity of astrocytes
67. Harvest and wash cells for flow cytometry
SECTION: Flow Cytometry to confirm purity of astrocytes
67.1. Aspirate media from T75 flask
SECTION: Flow Cytometry to confirm purity of astrocytes
67.2. Rinse plate with 10 mL DPBS
SECTION: Flow Cytometry to confirm purity of astrocytes
67.3. Aspirate DPBS
SECTION: Flow Cytometry to confirm purity of astrocytes
67.4. Add 2 mL 37ºC 1x trypsin EDTA (0.25%)
SECTION: Flow Cytometry to confirm purity of astrocytes
67.5. Incubate for 5 min in a 37°C, 95% air/5% CO2, 95% relative humidity incubator
SECTION: Flow Cytometry to confirm purity of astrocytes
67.6. Tap sides of flask to allow cells to detach
SECTION: Flow Cytometry to confirm purity of astrocytes
67.7. Add 10 mL FACS media (DPBS + 2% heat-inactivated horse serum)
SECTION: Flow Cytometry to confirm purity of astrocytes
67.8. Centrifuge at 4000 rcf at 4ºC, in a swinging bucket rotor for 10 minutes to pellet cells
SECTION: Flow Cytometry to confirm purity of astrocytes
67.9. Aspirate media
SECTION: Flow Cytometry to confirm purity of astrocytes
67.10. Resuspend in approximately 1 mL of fresh FACS media
SECTION: Flow Cytometry to confirm purity of astrocytes
67.11. Adjust cell number to a concentration of 1-5x106 cells/mL
SECTION: Flow Cytometry to confirm purity of astrocytes
67.12. Transfer 200 uL cell suspension to polystyrene round-bottom 12x75 mm falcon tubes
SECTION: Flow Cytometry to confirm purity of astrocytes
67.13. Centrifuge at 4000 rcf at 4ºC, in a swinging bucket rotor for 10 minutes to pellet cells
SECTION: Flow Cytometry to confirm purity of astrocytes
67.14. Add 200 uL/well paraformaldehyde (2%)
SECTION: Flow Cytometry to confirm purity of astrocytes
67.15. Incubate at room temperature for 15 min on shaker
SECTION: Flow Cytometry to confirm purity of astrocytes
67.16. Centrifuge at 4000 rcf for 5 min at 4ºC
SECTION: Flow Cytometry to confirm purity of astrocytes
67.17. Aspirate media using a fire-polished, cotton-plugged Pasteur pipette
SECTION: Flow Cytometry to confirm purity of astrocytes
67.18. Permeabilize (remove more cellular membrane lipids to allow large molecules like antibodies to get inside the cell easily) with 200 uL 0.05% Triton-X (in PBS)
SECTION: Flow Cytometry to confirm purity of astrocytes
67.19. Incubate at room temperature for 5 min on shaker
SECTION: Flow Cytometry to confirm purity of astrocytes
67.20. Centrifuge at 4000 rcf at 4ºC, in a swinging bucket rotor for 5 minutes to pellet cells
SECTION: Flow Cytometry to confirm purity of astrocytes
67.21. Aspirate media using a fire-polished, cotton-plugged Pasteur pipette
SECTION: Flow Cytometry to confirm purity of astrocytes
68. Blocking the non-specific binding of the staining antibody reagents
SECTION: Flow Cytometry to confirm purity of astrocytes
68.1. Prepare block solution:  1% donkey serum, 1% goat serum, 1x PBS; 490 μL PBS + 5 μL donkey serum + 5 μL goat serum
SECTION: Flow Cytometry to confirm purity of astrocytes
68.2. Add 100 μL blocking solution to each washed tube
SECTION: Flow Cytometry to confirm purity of astrocytes
68.3. Block for 20 min on ice
SECTION: Flow Cytometry to confirm purity of astrocytes
68.4. Centrifuge at 4000 rcf at 4ºC, in a swinging bucket rotor for 5 minutes to pellet cells
SECTION: Flow Cytometry to confirm purity of astrocytes
68.5. Aspirate blocking solution using fire-polished, cotton-plugged Pasteur pipet
SECTION: Flow Cytometry to confirm purity of astrocytes
69. Addition of primary antibody
SECTION: Flow Cytometry to confirm purity of astrocytes
69.1. Add 0.1-10 μg/mL (100 uL) of each primary labeled antibody (we use primary antibody diluted 1:200 in blocking solution). 
SECTION: Flow Cytometry to confirm purity of astrocytes
69.2. Incubate primary antibody for at least 30 min at room temperature
SECTION: Flow Cytometry to confirm purity of astrocytes
69.3. Centrifuge at 4000 rcf at 4ºC, in a swinging bucket rotor for 5 minutes to pellet cells
SECTION: Flow Cytometry to confirm purity of astrocytes
69.4. Aspirate primary antibody solution using fire-polished, cotton-plugged Pasteur pipette
SECTION: Flow Cytometry to confirm purity of astrocytes
70. Addition of secondary antibody
SECTION: Flow Cytometry to confirm purity of astrocytes
70.1. Dilute secondary antibodies in blocking solution according to dilution according to dilution factor listed in materials.
SECTION: Flow Cytometry to confirm purity of astrocytes
70.2. Resuspend cells in secondary antibodies (100 uL each)
SECTION: Flow Cytometry to confirm purity of astrocytes
70.3. Incubate for 1 hour at room temperature in the dark (cover with aluminum foil and turn lights off
SECTION: Flow Cytometry to confirm purity of astrocytes
71. Wash 3x
SECTION: Flow Cytometry to confirm purity of astrocytes
71.1. Centrifuge at 4000 rcf at 4ºC, in a swinging bucket rotor for 5 minutes to pellet cells
SECTION: Flow Cytometry to confirm purity of astrocytes
71.2. Aspirate antibodies using fire polished, cotton-plugged Pasteur pipette
SECTION: Flow Cytometry to confirm purity of astrocytes
71.3. Resuspend in 200 μL of ice-cold FACS media
SECTION: Flow Cytometry to confirm purity of astrocytes
72. Analyze on flow cytometer as soon as possible. Flow cytometry data analysis will involve identifying a population of cells through subsequent steps called gating and subjecting that population to various parameters through subsequent gating steps.
SECTION: Flow Cytometry to confirm purity of astrocytes
72.1. First, make forward scatter (FSC-A) and side scatter (SSC-A) density plots for the cell events using the BD Accuri C6 Software. The first gate will be applied to remove debris (dense accumulation of events on the bottom left corner). Thus, the gate will select all events measured by the flow cytometer except the debris, these events should all therefore be cells.
SECTION: Flow Cytometry to confirm purity of astrocytes
72.2. In the subsequent density plot of FSC-A x Width, the single cells are gated and selected, removing any clumps of cells from analysis.
SECTION: Flow Cytometry to confirm purity of astrocytes
72.3. Followed by this, the selected cells are analyzed for each of the cell-specific markers. First, microglia-specific marker Iba-1 was assessed by selecting an appropriate channel on the flow cytometer that is suited for the goat anti-rabbit 488 antibody detection. The Iba-1 positive cells are expected to be seen in the top left quadrant of the FSC-A x Iba-1 density plot.
SECTION: Flow Cytometry to confirm purity of astrocytes
72.4. Similarly, in the FSC-A x GFAP plot, GFAP-positive cells (astrocytes) are expected in the top left quadrant.
SECTION: Flow Cytometry to confirm purity of astrocytes
72.5. By combining the gates from steps 72.3 and 72.4, both cell populations can be viewed on the same plot. Gates are applied as shown to determine astrocyte purity.
  

SECTION: Confirm sex of pups
27.8. When solidified, remove the combs carefully without damaging the gel.
SECTION: Confirm sex of pups
27.9. Orient the gel in the gel electrophoresis apparatus such that the combs are placed towards the cathode. The DNA will be run from cathode to anode.