Glial Cell Culture Protocol (Originally from the Worley’s lab) Reagents 1. 2. 3. 4. 5. 6. 7. 8. 9. HBSS P/S/G 2.5%Trypsin MEM Horse Serum DNase / Sigma / DN-25 40 um Cell strainer (BD) 1X Trypsin Collagen or 1% Gelatin (the latter is much cheaper and gives similar results) 10. Corning or TPP Dishes Reagents and Media Preparation 1. Dnase: 100 mg Dnase 10 ml HBSS Filter Sterilization Store at -20oC in 1ml and 0.5 ml Aliquots 2. 10% Glia Media 500 ml MEM 55 ml Horse Serum 5 ml Pen/Strep/Glutamine 3. Collagen Coating Dilute Collagen with water 1:2 (e.g. 1 ml of Col and 2 ml of H2O) NOTE: Culturing Banker Style neurons requires 60 mm plates. For Mixed cultures where glia conditioned media is required only for feeding, flasks are sufficient. Coating Collagen Plates (Enough for 2 weeks) Preparation ** Only use Corning Brand 60 mm dishes. DO NOT USE ANY OTHER BRAND. 1. Collect 6 X medium flasks and 60 X 60 mm plates. This is enough for 2 weeks of culturing glia. (60 mm plates only for Banker Style) 2. Prepare Collagen Mixture (1 part collagen: 2 parts H2O). or 1% Gelatin in MilliQ water 10 ml total is enough. Plating ** Use Corning brand 60 mm dishes or TPP they have yielded best results. 1. Flash coat the flasks and plates by adding collagen mixture and swirling to completely coat plastic. 2. Remove collagen and reuse on next flask or plate until all flasks/plates have been flash coated. 3. Remove flash caps and plate tops and allow to dry O/N in hood. 4. The next morning elevate plates and flasks and treat with UV for 20-30 minutes. 5. Replace caps and tops and wrap in aluminum foil to protect from light. 6. Treated flasks and plates can be stored at 4o for 2 weeks. Glia Cell Culture (should be performed Monday or Tuesday) A. Glia Cell Culture (Should be performed Monday or Tuesday) 1. 2. 3. 4. Postnatal P1-3 day rats or P4-P5 mice Soak dissection instruments in 70% ethanol for 30 min before dissection. Warm 10% Glia Medium to 37oC in water bath. Warm up DNase (1 ml and 0.5 ml tube) and Trypsin (2X 1ml tube) at RT. B. Dissection (Keep all dissected tissue on ice) 1. 2. 3. 4. 5. Remove postnatal head into 60 mm Petri Dish with 2 ml HBSS Remove brain stem, cerebellum, and diencephalons. Peel off meningis and put cortex into a 60 mm dish with 4 ml HBSS on ice. 3-4 pups should be enough. After dissection, chop cortex into small pieces. ALL dissection should be done under a dissection microscope. Dissection should be performed as quickly as possible. Keep all dissected tissue on ice as much as possible. C. Digestion: 1. Transfer the chopped tissue in 4 ml HBSS into 15 ml conical Tube. 2. Add another 4 ml of HBSS into the above tube. The total volume should be 8 ml. 3. Add 1 ml of 2.5 % Trypsin and 1 ml Dnase into the tube, the total volume should be 10 ml, then mix. 4. Incubate at 37oC water bath for 15 min, mix every 5 min. 5. Transfer supernatant into a 50 ml conical tube (about 8 ml). 6. Add equal volume of 10% Glia Medium to block digestion 7. Add 5 ml HBSS, 0.5ml DNase, and 1 ml 2.5 % trypsin into 15 ml conical tube containing remaining digested tissue. 8. Incubate at 37oC water bath for 15 min, mix every 5 min. 9. Transfer the supernatant into the 50 ml conical tube (Step 5). 10. Add equal volume 10% Glia Medium into the 50 ml conical tube to block digestion. 11. Filter the cell suspension with a 40 um filter into another 50 ml conical tube. 12. Centrifuge at RT for 5 min, set speed at 3. Plating Cells: Day 1: . 1. Discard supernatant carefully. 2. Resuspend cell pellet in 4 ml 10% Glia Medium, and mix well. 3. Count cells and calculate density. Use Trypan Blue staining to count live cells. 4 parts stain: 1 part cells. Count only clear cells. 4. Plate 2x106 cells per collagen treated 75cm3 flask (PLATE 2X FLASKS PER DISSECTION) (Vol = ~15 ml). Day 2-4: 1. Before feeding, knock the flask firmly with the palm of your hand 3-4 times. This will knock off unwanted cells (microglia, neurons, etc). 2. Feed Cells daily with 10% Glia Medium. Day 7: 1. 2. 3. 4. 5. 6. 7. Remove media from flask and wash with PBS. Add 2.5 ml of TE. Resuspend in 10% Glia Midium and spin down 5 minutes at setting 2. Remove media. Be careful, pellet will be small. Resuspend in 10 ml of 10% Glia Medium Count Cells Plate cells on collagen coated flasks and plates: a. 20X 60 mm plates at 1.0 x 105 cells/plate in 4 mls (2.5 x 104cells/ml). (Only for Banker Style) b. 2X Medium sized flasks at 5 x 105 cells/flask in 15 mls. Day 8-10: 1. Feed cells daily with 10% Glia Medium. Day 11: 1. 30X 60 mm plates ready to use (Only for Banker Style). 2. Continue to let glia in flasks grow. Day 14-18: Sun Mon Tue Wed Thr Fri Dissection Seeding on flask Tapping and feeding the flask Tapping and feeding the flask Tapping and feeding the flask Split the flask To 25 dishes Feeding the dishes Feeding the dishes Feeding the dishes Ready to use Split the flask To 25 dishes Feeding the dishes Feeding the dishes Feeding the dishes Ready to use To 2 flasks Tapping and feeding the flask Tapping and feeding the flask Tapping and feeding the flask Tapping and feeding the flask Dissection Seeding on flask Tapping and feeding the flask Tapping and feeding the flask Tapping and feeding the flask Feeding the dishes Feeding the dishes Feeding the dishes Ready to use Split the flask To 25 dishes Sat 1. Split Flask into 30X 60 mm plates (1x105 cells/plate) (Only for Banker Style) and continue to feed until ready to use. * Dissections will be performed every other week. Therefore, there will be overlaps in plating cell protocols from every dissection (see calender for clarification).