Aspartate Uptake Experiment

3H D-Aspartate Uptake Experiment

Ray Swanson’s Lab – Ari Berman

1) Select plates with DIV 20 – 30 astrocytes. Perform steps 2 – 5e in 37C water bath. The remaining steps at RT. 2) Wash out media 1X with BSS-P (BSS with 5mM PIPES) containing 2mM glucose 3) Add 280L BSS-P in each well 4) Add 20L of aspartate cocktail to each well 5) Assess uptake in the following intervals (2, 4, 6, 8, 12, and 15 min) a. Plan out intervals in a manner that allows the differing uptake times. (see last page) b. Aspirate uptake medium c. Wash 3X with 400L ice-cold HBSS-A d. Add 400L lysis buffer to each well to lyse the cells (use 1mL pipettor for this, not repeater pipettor) e. Once all cells have lysis buffer added, scrape wells with pipette tip to ensure complete lysis. f. Incubate lysis buffer for 10 minutes at RT. 6) Add 350L from each well containing lysate to 5mL scintillation cocktail and count for 3H for 5 min using precision % 2 = 2 (user #5). 7) Add 20L of the uptake cocktail to one scintillation vial for baseline counts, for conversion of the counts to uptake amount. 8) Measure protein content from 30L lysate using the BCA kit. Duplicate each measurement, 15L each.

· Aspartate uptake cocktail (final - 100M cold D-Aspartic Acid and 27nM D- Aspartate-[2,3-3H] (0.04Ci/well; 270pmol/Ci; 1Ci/L)) o 66.55mg cold D-Aspartic Acid in 50mL BSS-P – 10mM stock solution o 75L (1.5mM) cold 10mM D-Aspartic Acid o 1L D-Aspartic Acid-[2,3-3H] (1Ci) (or BSS-P for control cocktail) o 424L BSS-P · Lysis buffer o 0.1N NaOH + 0.01% SDS (200L of 10N NaOH, 20L 10% SDS, 20mL ddH2O) – make fresh, per each 24 well plate. · BSS-P Solution o NaCl (140mM): 8.2g o KCl (3.1mM): 231mg o CaCl2•2H2O (1.2mM): 176mg o MgSO4•7H2O (1.2mM): 296mg o KH2PO4 (0.5mM): 68.1mg o Glucose (2mM): 360.32mg o PIPES (5mM): 1.512g (Crush into fine powder before adding to the flask) o Fill to 1L with ddH2O o Equilibrate to pH7.2 with 10N NaOH – PIPES will dissolve as pH approaches 7.2. If PIPES remains undissolved, sonicate the solution for 2min to break up aggregates then stir and re-pH to 7.2. o Add 1/2mM (29.3mg) NaCl until 280 – 320mOsm is reached · HBSS-A o 1 500mL bottle of Hank’s BSS without Ca++ or Mg++ salts o Remove 17.5mL from the bottle and discard o Add 5mL 1M HEPES from US Cell Culture (1:100 dilution) to bottle o Add 12.5mL Pen/Strep from UC Cell Culture (2.5X) to bottle o Mix and filter enough for experiment. Stock lasts 1month at 4C.

Administration strategy for uptake assay

A B C D E F Wash 10:45 10:30 10:15 12:00 11:45 11:30 1 2:00 2:00 2:00 4:00 4:00 4:00 Uptake Time Administer 8:45 8:30 8:15 8:00 7:45 7:30 Wash 13:00 12:45 12:30 14:15 14:00 13:45 2 6:00 6:00 6:00 8:00 8:00 8:00 Uptake Time Administer 7:00 6:45 6:30 6:15 6:00 5:45 Wash 17:15 17:00 16:45 19:30 19:15 19:00 3 12:00 12:00 12:00 15:00 15:00 15:00 Uptake Time Administer 5:15 5:00 4:45 4:30 4:15 4:00 Wash 2:30 2:15 2:00 18:45 18:30 18:15 Controls - 4 2:00 2:00 2:00 15:00 15:00 15:00 Uptake Time Administer 0:30 0:15 0:00 3:45 3:30 3:15 Start Interval: 0:15

- Bottom time indicate wall clock time from first administration of cocktail to wells - Middle time indicates the uptake time for that well - Top time indicates the wall clock time at which to wash out the uptake cocktail - Start this experiment with well C-4, follow the times! - Row 4 consists of negative controls. They are given the control cocktail as outlined above