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1 Primary Culture Information Packet McLaughlin Lab So, you or your lab is interested in learning primary cultures—Here is what we can do for you. 1. Attached are protocols for our plate prep, dissection/dissociation, media formulations, and culture care instructions. 2. You are welcome to watch 2 dissections in our lab, performed by our expert staff, provided you schedule 2 weeks in advance. 3. Once our cells are plated, you may look at them twice during their maturation. We suggest that you already have an IACUC protocol in place for the animals you will be sacrificing. One week after watching our dissection, it would be wise to perform a dissection/dissociation on your own while the information is still fresh. That way, when watch another dissection in our lab, you can trouble shoot your methods and techniques. Contact Information Lab Phone: 936-3872 Location: 8141A MRB III 2 COVERSLIP CLEANING 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. In a 500mL beaker, place 2 boxes of small coverslips, or one large box of coverslips, and add 250 mL of Milli Q water. Swirl gently for 2 minutes Decant the water into the sink, make sure not to let any coverslips pour out Add 250 mL of new Milli Q water Sonicate for 15 minutes Repeat steps 1-5 Decant the water, and add 250 mL of 0.1 N HCl a. 248 mL of Milli Q water b. 2 mL of 12 N HCl Cover the beaker with parafilm, and place the beaker on the orbital shaker for 1 hour Decant the 0.1 N HCl into the appropriate hazardous waste drum Wash 3 times with Milli Q water Add 250 mL of 95% ethyl alcohol, and place the beaker on the orbital shaker for 1 hour Decant the 95% ethyl alcohol into the appropriate hazardous waste drum Wash 2 times with Milli Q water in the hood Add 250 mL of 95% ethyl alcohol, and place parafilm on the top. Store in the cell culture room until use 3 Dissociation for Neuronal Culture One Day Before Dissection 1. UV culture plates for 1 hr. 2. Make Borate Buffer/Poly-L-Ornithine [sigma, P-3655] (2 mL: 1 mg) to coat plates with and without coverslips. 3. Add 2 ml of PLO to each well, and leave it on for at least 1 hr. 4. Wash each well 2X with tissue culture water. Remove lids and allow it dry in the hood for 2-4 hrs. 5. Add 1.5 ml of plating media to each well, and place plates in the incubator until dissection. Before Dissection 1. Weight out 3 mg of Trypsin [sigma, T9935] and dissolve in 5 mL of HBSS. Sterile filter into a 35 mm culture dish & label. 2. Add 3 mL of HBSS to 5 of the 35 mm culture dishes. 3. Add 450 µL of Trypan blue to a 1.5 mL eppendorf tube. 4. Prepare 70% alcohol, scissors, forceps, and absorbent pads for dissection. 5. Use syringe with an 18 gage needle to remove sedative from the bottle; then, replace the 18 gage needle with a 22 or 25 gage needle for injection into the rat. 6. Calculate the number of mL that are need to plate all the wells. 7. Place plating media into water bath at 37ºC. Dissection 1. Inject the rat with the sedative, Nebutol, by IP injection, then wait a few minutes for her to relax and lay down 2. Check her “blink” reaction and squeeze her foot to determine if she is sedated enough. 3. Place her on the absorbent pad belly up, and spray the belly with 70% alcohol. 4. Use the large forceps to lift the belly skin. Cut open with the heavy-duty dissecting scissors. Cut the skin along the midline up to the diaphragm area. 5. Then cut the muscle along the midline to expose the embryos and the diaphragm. 6. Cut both sides of the rat’s diaphragm to stop respiration. 7. Use the large forceps to pull all the embryos. Cut the embryos open with fine-tip dissecting scissors. Cut off the head of the embryo into HBSS culture dish. 8. Transfer one embryo head into a new dish of HBSS that is now under the microscope. 9. Hold the head straight up. Puncture the skin and membrane outside of the brain with the dissecting forceps. Peel the skin and membrane toward the front of the head until the brain pops out. Turn the head around and peel toward the back of the head until the brain stem is exposed. Put the dissecting forceps under the brain stem and pull toward the back of the head until the brain is separated with the skull. Pinch-cut the brain stem to separate the brain completely. 10. Place the brain in a new HBSS dish. With the dorsal of the brain facing up, hold the brain stem gently with forceps. Separate the hemispheres with the dissecting forceps. Pull the cerebral cortex away from the brain stem. 11. Place one cortex in a new dish of HBSS. Flip the cerebral cortex so that the outside is facing up. Pinch-cut the olfactory bulb with the dissecting forceps. Peel the meninges from the front toward the back. Try to peel it off in one piece. 12. Pinch cut any mid brain structures underlying the cortex. 13. Transfer the cortex to a new dish with HBSS. Repeat for the other cortices. 4 NOTE: Dissection time should take less than 30 mins!!! 14. Transfer the dish with all the cortices to the hood. Transfer the cortices into Trypsin solution with forceps, making sure to take as little HBSS as possible with them. 15. Let the cortices sit in Trypsin for 30 mins in the hood at RT. During Trypsinization 1. Aspirate the coating media from the culture plates. 2. Add 3 mL of HBSS into two more 35 mm culture dishes for wash. 3. Add 5 mL of plating media into two 15 mL tubes. Set out 3 more tubes for titration. After Trypsinization 1. Transfer the cortices form the Trypsin to a new dish with HBSS with a Pasteur pipette, one at a time. Do not break the cortices. Swish them around to wash off the Trypsin. Repeat 2. Transfer the cortices with a new Pasteur pipette to the first tube of plating media. Slowly triturate the cortices with the Pasteur pipette to maximize the total cell number & viability. Let the chunks settle down to the bottom of the tube. 3. Transfer ONLY the cell suspension (not the chunks) to a new 15 mL tube with a Pasteur pipette. Again, slowly triturate with cortices. Continue this process until the suspension looks even. 4. When Titrations are complete, add 50 µL of the cell suspension to the eppendorf tube with Trypan blue (a 1:10 dilution). Mix well by flipping the tube upside-down several times. 5. Remove 50 µL of Trypan blue mixture, and add it to the hemocytometer (it takes approximately 15-20 µL to fill each side). 6. Count the number of live cells and dead cells in 3 of the 4X4 quadrants (1 on one side, & 2 on the other side). 7. Use this series of formulas to determine the needed cell to media dilution. a. (Live Cells)/(3 squares) = y b. y * 10 = z (for 1:10 Dilution in Trypan blue) c. z * 10,000 = (hemocytometer volume correction) 8. Dilute the cell suspension to the desire plating density with proper media a. 335,000 cell/mL 9. Plate 2 mL/well, and change the pipette after every 2 plates. 10. Swirl media bottle to insure proper homogeneity of cell suspension in between plates. 11. Make sure to mark the date on the plate cover. 12. Carefully return dishes to the incubator, making not to splash media on lids or disrupt cells. 5 Maintance of Primary Cultures Primary cultures are maintained at 37oC & 5% CO2. Media is partially replaced every 2 – 3 days. For Neuronal Cultures: Glial cell proliferation is stopped by adding 1-2µM cytosine arabinoside to the cultures two days after dissociation. On the third day, the media is changed from Plating Media to Neurobasal. All Neuronal experiments are conducted 3 weeks after dissociation. For Mixed Cultures: Glial cells are inhibited after two weeks in culture with 1-2 M cytosine arabinoside, after which the cultures were maintained in growth medium containing 2% serum and without F12-nutrients. All experiments are conducted one week following mitotic inhibition (25-29 days in vitro). 6 Borate Buffer (4º) 4.76 g Boric Acid 2.54 g Borax 1000 mL of ddH2O pH to 8.4 with NaOH Sterile Filter Plating Media (4º) 40 mL F12 40 mL FBS 420 mL Dulbecco Modified Eagle Medium (DMEM) 1 mL of PS 200 uL of 200 mM L-Glutamine D2C (4º) 10 mL FBS 1 mL PS 12.5 mL of 1M hepes 471.5 mL Dulbecco Modified Eagle Medium (DMEM) 6.25 mL of 200 mM L-Glutamine Neurobasal (4º) 1 bottle (500 mL) Neurobasal Media 1 bottle (10 mL) 50X B27 P/S Stock Ara-C (500X) (-20º) 100 mg Ara-C 6 mL sterile H2O Sterile Filter Aliquot 0.5 mL Working Ara-C (4º) 20µL Stock 10 mL sterile ddH2O 10% Formaldehyde (RT) 5 mL of 37% Stock Solution 45 mL of PBS Glucose Free Balanced Salt Solution 5.26 g NaCl 0.125 g KCl 66.6 mg CaCl2 1.43 g Hepes 600 mL ddH2O Sterile Filter pH 7.3 PLO 1 mg PLO : 2 mL Borate Buffer Sterile filter Make up fresh each time it is used HBSS (4º) 8.4 g NaCl 224 mg KCl 2.38 g Hepes 1.0 g Glucose 1000 mL Milli Q pH to 7.3 with NaOH Sterile Filter Roccal II (10%) (RT) 10% Benzalkonium Chloride (B1383 Sigma) 1.25% Ethyl Alcohol Dissolve in alcohol with stirring Add 88.75% Milli Q water MEM/BSA/Hepes 487.5 mL of clear MEM 12.5 mL of 1 M Hepes 50 mg of BSA Sterile Filter 7 Tissue Culture Media & Supplements Product DMEM w/o L-glutamine DMEM w/ L-glutamine MEM w/o L-glutamine Neurobasal Opti-Mem B27 Supplement N2 Supplement Pen/Strep L-Glutamine FBS Cytosine Arabino Furanoside (Ara-c) Poly-L-ornithine F-12 Nutrient Ham Hepes Buffer Vendor Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Cambrex Hyclone Catalog # 11960-089 CASE 11995-073 CASE 51200-038 21103-049 11058-021 17504-044 17502-048 15140-122 17-605C SH30070.03 Sigma Sigma Sigma Sigma C-6645 P-3655 N4888 H0887