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 32 34 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 151.25 NaCl 130 58.44277 3.7987800 7.5975601 Cl 138 5 KCl 3 74.5513 0.1118269 0.2236539 K 3 5 NaH2PO4 1.25 120 0.075 0.15 PO4 1.25 Glucose 10 180.16 0.9008 1.8016 HCO3 20 NaHCO3 20 84.01 0.8401 1.6802 SO4 1.3 MgSO4 1.3 246.5 0.160225 0.32045 Mg 1.3 CaCl2 *2H20 2.5 147 0.18375 0.3675 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 10 180.2 0.901 1.802 Cl 145.1 NaCl 136 58.44277 3.9741083 7.9482167 K 2.5 6 2 KCl 2.5 74.5513 0.0931891 25 0.1863782 5 Mg 1.3 MgCl2 1.3 203.3 0.132145 0.26429 Ca 2 HEPES 10 238.31 1.19155 2.3831 Glucose 10 CaCl2 (add 2 147 0.147 0.294 HEPES 10
last) choline cutting soln Conc. (mm) M.W. G/250 ml G/L G/ 2L bicarb 20 84.01 1.6802 3.3604 NaPO4 (1 1.25 138 0.1725 0.345 H20) KCl 3 74.56 0.22368 0.44736 MgSO4 1.3 120.363 0.1564719 0.3129438 Glucose 10 180.2 1.802 3.604 add in small batches CaCl2 0.5 147 0.0735 0.147 choline 110 139.6 3.839 15.356 chloride ascorbic 11.6 198.1 0.57449 2.29796 acid pyruvic acid 3.1 110 0.08525 0.341 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 3.5 74.56 0.52192 2.61 GLUCOSE 10 180.2 3.604 18.02 NaCl 126 58.44 14.72688 73.63 NaH2PO4* 1.25 137.99 0.344975 1.72 MgSO4*7 0.5 246.48 0.24648 1.23 CaCl2*2H2 O 1 147.02 0.29404 1.47 bicarb (from other stock) 26 84.01 - - 2.18426 shielding ACSF
Salt mm MW G/L KCl 2.5 74.56 1.86 GLUCOSE 10 180.2 18.02 NaCl 126 58.44 73.63 NaH2PO4* 1.25 137.99 1.72 MgSO4*7 2 246.48 4.93 CaCl2*2H2 4 147.02 5.88 O bicarb (from other stock) 26 84.01 2.18 cutting saline mm MW G/2L- 1x G/L KCl 2.5 74.56 0.47 1.86 NaH2PO4* 1.25 137.99 0.43 1.72 MgSO4*7 10 246.48 6.16 24.65 CaCl2*2H2 O 0.5 147.02 0.18 0.74 alternate monobasic phosphate formulations mm MW G/2L- 1x G/L NaH2PO4* 1.25 137.99 0.43 1.72 NaH2PO4* 1.25 119.98 0.37 1.50-7- NaH2PO4 1.25 268.07 0.84 3.35
Regular ACSF (1L) Mol wt g ml [mm] NaCl 58.4 6.9496 119 KCl 74.6 0.1865 2.5 MgSO4.7H 2O 246.5 0.32045 1.3 CaCl2 1M 2.5 2.5 NaH2PO4 138 0.138 1 Na2HCO3 84 2.184 26 Glucose 180.2 1.9822 11 HPLC water to 1L Slicing solution (500 ml) 500ml Mol wt g ml [mm] g ml 2.5469168 KCl 74.6 0.19 9 0.10 MgSO4.7 246.5 1.73 7 0.86 NaH2PO4 138 0.14 1.0144927 54 0.07 Na2HCO3 84 2.2 26.190476 19 1.10 Glucose 180.2 1.98 10.987791 34 0.99 Sucrose 342.3 78.04 228 39.02 CaCl2 1M 0.5 0.5 0.25 HPLC water to 1L Use ice-cold
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 74.56 3.728 10 GLUCOSE 180.2 36.04 126 NaCl 58.44 147.25 1.25 NaH 2 PO 4 *H 2 O 137.99 3.45 1 MgSO 4 *7H 2 O 246.48 4.93 2 CaCl 2 *2H 2 O 147.02 5.88 2 L 10X BICARBONATE STOCK mm Component MW g 26 NaHCO 3 (Sodium Bicarbonate) 84.01 43.685
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 74.56 12.50 1.864 1.25 NaH 2 PO 4 *H 2 O 137.99 6.25 1.724 10 MgSO 4 *7H 2 O 246.48 50.00 24.648 0.5 CaCl 2 *2H 2 O 147.02 2.50 0.736 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) 180.2 9.92 234 Sucrose 342.3 400.5
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) 74.56 50 3.728 10 GLUCOSE 180.2 200 36.040 126 NaCl (Sodium Chloride) 58.44 2520 147.269 1.25 NaH 2 PO 4 *H 2 O (Sodium phosphate) 137.9 9 0.2 MgCl 2 *6H 2 O (Magnesium Chloride) 203.3 1 2 CaCl 2 *2H 2 O (Calcium Chloride) 147.0 2 g 25 3.450 4 0.813 8 5.881
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) 74.56 50 1.864 10 GLUCOSE 180.2 200 18.020 126 NaCl (Sodium Chloride) 58.44 2520 73.635 1.25 NaH 2 PO 4 *H 2 O (Sodium phosphate) 137.9 9 0.63 MgSO 4 *7H 2 O (Magnesium Sulfate) 246.4 8 2 CaCl 2 *2H 2 O (Calcium Chloride) 147.0 2 G 25 1.725 12.6 1.553 8 2.941 1 Liters 10 Times concentrated 1 Liter of 10X choline solution for perfusion & slicing (MINUS BICARB) mm Component MW ml 1M stock 110 Choline chloride 139.6 1100.00 153.560 10 Glucose 180.2 100.00 18.020 2.5 KCl (potassium chloride) 74.56 25.00 1.864 1.25 NaH 2 PO 4 *H 2 O (Sodium phosphate) 137.9 9 10 MgSO 4 *7H 2 O (Magnesium Sulfate) 246.4 8 0.5 CaCl 2 *2H 2 O (Calcium Chloride) 147.0 2 g 12.50 1.725 100.00 24.648 5.00 0.735
Excitability ACSF from Maffei, nat Neuro 2004 1L 10X Excitability ACSF mm Component MW G 3.5 KCl 74.56 2.61 10 GLUCOSE 180.2 18.02 126 NaCl 58.44 73.63 1.25 NaH 2 PO 4 *H 2 O 137.99 1.72 0.5 MgSO 4 *7H 2 O 246.48 1.23 1 CaCl 2 *2H 2 O 147.02 1.47 1L 10X Shielding ACSF mm Component MW G 2.5 KCl 74.56 1.86 10 GLUCOSE 180.2 18.02 126 NaCl 58.44 73.63 1.25 NaH 2 PO 4 *H 2 O 137.99 1.72 2 MgSO 4 *7H 2 O 246.48 4.93 4 CaCl 2 *2H 2 O 147.02 5.88
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 2 246.48 1.46 2 CaCl 2 *2H 2 O 147.02 2.12