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Edited by Barbara Gibbons (2004). Adapted from protocol used for immunofluorescent detection in the lab of Dr. Santosh D’Mello, Molecular Cell Biology UTD (1999). INTRODUCTION Neural tissue extractions can be studied as living organotypes such as slices, or as dissociated cells that can be cultured for recording. Fixation processed dissociated cells are commonly used for localization of proteins and other macromolecules involved insignalingand metabolism. During the dissociation procedure, many of the processes are lost along with the connective tissue. However, some dendrites remain intact, and limited dendritic and axonal growth can reestablish in culture. Unlike cell lines that continue to multiply, healthy neurons do not undergo further division and have limited viability. Glial cells do continue to proliferate, and can quickly overgrow and compromise the value of the neuron population. To prevent glial overgrowth, the culture or plated cells are treated with an anti-mitotic agent to prevent replication of glia. Dish cultures, maintained by regularly changing the medium, provide a unique recording preparation that continue to be viable for up to one week. For immunostaining purposes, the cells are cultured briefly in wells containing pretreated plates that the cells adhere to. The plated cells can then be easily manipulated for immunoprocessing. MATERIALS Poly-d-Lysine treated plates(sterile round glass coverslips) Dish with wells size appropriate for plates BSA (Bovine Serum Albumin) HEPES MgSO4 Glutamine Gentamycin FCS(Fetal Culture Serum) ARA-C anti-mitotic agent Ethanol Trypsin Trypsin inhibitor DNAse 0.1M CaCl2 4% paraformaldehyde in 0.1M PBS 0.2M PBS (diluted from 10X stock) NaCl KCl 125 ml flask 1 L flask 2 L flask 50 ml centrifuge tubes with holder 15 ml centrifuge tubes with holder 1 ml centrifuge tube Ridged Erlenmeyer flask Pipette(low µ) Pipette for transfer of dissociation solutions (1-20 ml) Cotton-plugged pasteur pipette Syringe 2 large filters Balance Centrifuge Orbital shaker Hemocytometer SOLUTIONS 1.KREBS buffer stock 10X • Measure out and add to a 2 L flask the following: 2.237 g KCl 1.565 g MgSO4(anhydrous) 1.711 g NaH2PO4•7H2O •Add dH2O to 1 L mark and stir on stir plate. 2.Serum •Add 10 ml KREBS to 125 ml flask and dilute with dH2O to 100 ml mark. • To the diluted KREBS add: 300 mg BSA 100 µl 1.2M MgSO4. 1ml 1M HEPES. • Let stand for 10-15 min with occasional mixing. *** Be careful not to agitate the flask too much; the albumin has a tendency to stick to the glass and not dissolve.*** 3. 0.1M PBS • To 250 ml of 0.2M PBS add: 43.8 g NaCl 1 g KCl •Mix well and add dH2O to a final volume of 500 ml. 4. Dissociation solutions •Label three 50 ml centrifuge tubes: #1, #2, #3. •Label two 15 ml centrifuge tubes: #4, #5. •Add to the tubes the following ingredients in this order: •Let solutions stand in capped tubes for 15 min or more with frequent gentle mixing. •Then add 4ml of solution #4 to solution #3. •Filtering: Underthe hood, with a syringe and two large filters, remove plunger and hook filter onto bottom of syringe. Filter solutions in numerical order, changing filter after #3. ***Solutions can be frozen (-20ºC) after this step for later use.*** PROCEDURE 6.Meanwhile, make theculture medium(≈ 180 ml final volume): • Add 1 ml gentamycin and 5 ml glutamine to pre-made serum, and warm. • Warm 3.6 ml KCl. • Then add 20 ml FCS (≈10%) and the warm 3.6 ml KCl (18 µl per ml) to the serum mix, and set aside. ***You want to see white cells in medium, not a tornado. If tornado, titrate 6 ml of solution #4 into tube with pipette, and spin again for 2 to 3 min.*** 8.Gently remove top layer (supernatant) without disturbing pellet. Add 6 ml solution #4 to tube (with pellet), and transfer to fresh 15 ml tube. Suction and release gently 25 times with cotton-plugged pasteur pipette to separate cells. Then place top on tube, and allow to stand for 5 min. ***The top (80%, 80%,95%) of mixture removed in the next steps now contain the cells separated from connective tissue and other tissue debris.*** 9.Carefully transfer upper 80% (≈ 4-5 ml) to fresh 50ml tube. Retain 15 ml tube containing the remaining 20%. 10.Add 4 ml of solution #4 to 15 ml tube with remaining 20%. Repeat suction – release separation 25 times. Leave 10 min with cap on. 11.Transfer the upper 80% of this tube’s contents to the 50 ml tube holding the first 80% transferred in step #8. Total volume should be about 8 ml. Add 10 ml of solution #5 and mix. Let stand for 5 min. 12.Then transfer the top 95% to a 50 ml tube. There should not be a pellet at this point. Spin at 4000 rpm for 10 min, and discard supernatant. Remaining in the tube are suspended dissociated cells. 13.Heatculture medium(from step 6) to 37ºC. 14.Add the content of the tube (suspended dissociated cells) to 20 ml ofculture medium. Pipette suction 10 – 15 times to mix and separate clumps. Remember no bubbles. This is thecell-mix. 15.Aliquot 450 µl ofculture mediuminto 1 ml tube. Pipette 50 µl ofcell-mixinto tube ofculture medium, and mix well. This is a counting sample of the final cell suspension.TUBE #1 TUBE #2 TUBE #3 TUBE #4 TUBE #5 25 ml KREBS 25 ml KREBS 21 ml KREBS 15 ml KREBS 12.5 ml KREBS 9-10 mg Trypsin 2.1 mg DNAse 1.2 mg DNAse 12.5 µl MgSO4 21 µg MgSO4 7.8 mg Trypsin inh. 12.5 µl CaCl2 19 µg MgSO4