Glucose Cl NaCl K 2.5

Transcription

1 Brains were removed and immersed in ice- cold solution containing the following (in mm): 87 NaCl, 2.5 KCl, 1.25NaH2PO4, 7MgSO4, 0.5 CaCl2, 25 NaHCO3, 25 glucose, and 75 sucrose, ph 7.4, 315 mosm. Parasagittal cortical slices (300 um, 30 angle) were obtained by using standard procedures (Galarreta et al., 2008). Initially, slices were incubated at C for 25 min in the same solution. Subsequently, the slices were maintained for at least 25 min at room temperature in an oxygenated solution containing he following (in mm): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, 26 NaHCO3, 20 glucose, 4 lactic acid, 2 pyruvic acid, and 0.4 ascorbic acid, ph 7.4, 315 mosm [artificial CSF (acsf)] Bicarb ACSF (from pk/lab) Salt Conc. (mm) Ion concentrations (mm) M.W. G/500 ml G/L Na NaCl Cl KCl K 3 5 NaH2PO PO Glucose HCO3 20 NaHCO SO4 1.3 MgSO Mg 1.3 CaCl2 *2H Ca 2.5 Glucose 10 Culture ACSF (from burrone & murthy) Salt Conc. (mm) Ion concentrations (mm) M.W. G/500 ml G/L Na 136 Glucose Cl NaCl K KCl Mg 1.3 MgCl Ca 2 HEPES Glucose 10 CaCl2 (add HEPES 10

2 last) choline cutting soln Conc. (mm) M.W. G/250 ml G/L G/ 2L bicarb NaPO4 ( H20) KCl MgSO Glucose add in small batches CaCl choline chloride ascorbic acid pyruvic acid modified from pk's ACSF make up stock from first 5 ingredients, store at 4 C add last 4 to smaller aliquote excitable ACSF Salt mm MW G/2L- 1x G/L - 10X KCl GLUCOSE NaCl NaH2PO4* MgSO4* CaCl2*2H2 O bicarb (from other stock) shielding ACSF

3 Salt mm MW G/L KCl GLUCOSE NaCl NaH2PO4* MgSO4* CaCl2*2H O bicarb (from other stock) cutting saline mm MW G/2L- 1x G/L KCl NaH2PO4* MgSO4* CaCl2*2H2 O alternate monobasic phosphate formulations mm MW G/2L- 1x G/L NaH2PO4* NaH2PO4* NaH2PO

4 Regular ACSF (1L) Mol wt g ml [mm] NaCl KCl MgSO4.7H 2O CaCl2 1M NaH2PO Na2HCO Glucose HPLC water to 1L Slicing solution (500 ml) 500ml Mol wt g ml [mm] g ml KCl MgSO NaH2PO Na2HCO Glucose Sucrose CaCl2 1M HPLC water to 1L Use ice-cold

5 These recipes come as handy labels to tape on your ACSF bottles.. To make saline, add ~700 ml of milliq water into a 1 liter bottle, and then add 100 ml (a 100 ml graduated cylinder is plenty good enough precision) of each of the following. Mix after each of the two additions. Then pour into a 1 liter volumetric and bring it up to the final volume with milliq water.. 2 L 10X ACSF (with 1mM Mg NO BICARB) (291 mosm) mm Component MW G 2.5 KCl GLUCOSE NaCl NaH 2 PO 4 *H 2 O MgSO 4 *7H 2 O CaCl 2 *2H 2 O L 10X BICARBONATE STOCK mm Component MW g 26 NaHCO 3 (Sodium Bicarbonate)

6 To make sucrose slicing solution saline, add ~100 ml of milliq water into a 250 ml bottle, and then add 20 ml (a 25 ml graduated cylinder is plenty good enough precision) of each of the following, along with the 10x NaHCO 3 stock. Mix after each of the three additions. Then bring bring it up to 200 m1 with milliq water. 1L OF 10X Sucrose Saline (No Bicarb) mm Component MW ml 1M stock g 2.5 KCl NaH 2 PO 4 *H 2 O MgSO 4 *7H 2 O CaCl 2 *2H 2 O To make the 10x sucrose slicing solution, measure out 119 ml of milliq water into a 250 ml bottle, and place on heated a stir plate with a magnetic stir bar. Stir while adding the glucose first, wait for it to dissolve, and then the sucrose in parts, about 50g at a time, and let each part dissolve before adding the next. Watch carefully and don't let it overheat or you will cook the solution and obtain some kind of interesting caramel mixture! 500 ml 10X Sucrose Slicing Stock mm Component MW g 11 GLUCOSE (dissolve first) Sucrose

7 These solutions are used less frequently: 2 Liters 10 Times concentrated 2 LITERS OF 10X Low Mg (0.2 mm) STOCK (MINUS BICARB) mm Component MW ml 1M stock 2.5 KCl (potassium chloride) GLUCOSE NaCl (Sodium Chloride) NaH 2 PO 4 *H 2 O (Sodium phosphate) MgCl 2 *6H 2 O (Magnesium Chloride) CaCl 2 *2H 2 O (Calcium Chloride) g

8 1 Liters 10 Times concentrated 2 LITERS OF 10X Medium Mg (0.63 mm) STOCK (MINUS BICARB) mm Component MW ml 1M stock 2.5 KCl (potassium chloride) GLUCOSE NaCl (Sodium Chloride) NaH 2 PO 4 *H 2 O (Sodium phosphate) MgSO 4 *7H 2 O (Magnesium Sulfate) CaCl 2 *2H 2 O (Calcium Chloride) G Liters 10 Times concentrated 1 Liter of 10X choline solution for perfusion & slicing (MINUS BICARB) mm Component MW ml 1M stock 110 Choline chloride Glucose KCl (potassium chloride) NaH 2 PO 4 *H 2 O (Sodium phosphate) MgSO 4 *7H 2 O (Magnesium Sulfate) CaCl 2 *2H 2 O (Calcium Chloride) g

9 Excitability ACSF from Maffei, nat Neuro L 10X Excitability ACSF mm Component MW G 3.5 KCl GLUCOSE NaCl NaH 2 PO 4 *H 2 O MgSO 4 *7H 2 O CaCl 2 *2H 2 O L 10X Shielding ACSF mm Component MW G 2.5 KCl GLUCOSE NaCl NaH 2 PO 4 *H 2 O MgSO 4 *7H 2 O CaCl 2 *2H 2 O

10 200mL 36X Shielding additive add to regular ACSF to increased divalents 2x, Will maintain soln osmolarity, reduce [other] by < 3% 2.8 ml additive for 100 ml ACSF, 14 ml for 500 ml ACSF mm Component MW G 1 MgCl CaCl 2 *2H 2 O