Jacobs:Protocol OFF (Large Chambers)

Materials

 * Cells at ~80-90% confluence (plate ~250K cells/slide 2 days prior)
 * Tweezers
 * -MEM / 5% FBS / 5% CS
 * Pipette Bulbs
 * Tubing (1/8 ID x ¼ OD x 1/16 wall)
 * Cell Scraper
 * 1-mL pipettes (4)
 * Plastic adapters (4)
 * 5-mL syringes (8) and 10-mL syringes (2)
 * Timers (2)
 * 20-mL syringe (1)
 * Petri dishes
 * TriReagent or RIPA Lysis buffer and inhibitors
 * Waste beaker
 * Flow chambers: tops and bottoms (16)
 * Clamps (>8)
 * Screws soaked in 100% EtOH
 * Tape
 * Gaskets (15)
 * PBS
 * P200 or P100 and pipette tips
 * Electric Screwdriver
 * Microcentrifuge tubes (24 for mRNA, 6-12 for protein)
 * Scissors
 * Plastic buckets (2) with ice
 * Marker

Procedure
Set-up
 * 1) Lay down bench-top padding, warm media in water bath, turn on right computer, gray box (after turning on computer!), and flow meter.
 * 2) Set out big chamber tops and bottoms (16 of each), screws, gaskets (15), scissors, waste beaker, plastic bucket
 * 3) Cut tubing (1/8 ID x 1/4 OD x 1/16 wall):
 * 4) 4 long pieces (2 cabinet lengths)
 * 5) 4 medium pieces (6 in)
 * 6) 32 short pieces (1 decimeter)
 * 7) Attach two short pieces to each chamber
 * 8) Break 4x 1-mL pipettes and discard tapered end; remove cotton with tweezers and then use pipettes to attach long tubing to medium tubing
 * 9) Attach other end of long tubing to Hamilton syringes on pump
 * 10) Open Wintest
 * 11) Mover 1/2  Local  Switch from Off to High
 * 12) Mover 1/2  Waveform  900 cycles = 15 min (7200 c = 2 hr); Amp ± 4.2 mm
 * 13) Put padding (3 large pieces) in incubator
 * 14) Insert flow meter into tubing
 * 15) Pour media into ~6 Falcon tubes
 * 16) Remove locking mechanism from 8x 5-mL syringes and 2x 10-mL syringes and fill with media (from 50-mL conical tubes)
 * 17) Get 2 pipette bulbs

Set-up: Chambers
 * 1) Remove 8 slides from incubator in cell culture room
 * 2) Attach a 5-mL syringe to one chamber tubing; tilt chamber upward to minimize air bubbles; fill tubing with media and spread over chamber top with bulb (do not let media touch inlet at opposite end of chamber)
 * 3) Attach a 10-mL syringe to second inlet tubing and fill with media; smooth over top of chamber with bulb
 * 4) Remove any air bubbles from media using a bulb
 * 5) Place slides cell-side-down on top of chamber
 * 6) Check that slide is in place using tweezers
 * 7) Remove excess media with bulb
 * 8) Put gasket down on top of slide (cover black rubber perimeter)
 * 9) Insert screws on opposite sides of chamber, 3-4 on each side; push down on chamber top while screwing into bottom of chamber; insert screws by hand and then use Allen wrench to tighten
 * 10) Tap chamber on bench (tubing upward) to remove air bubbles (don’t need to do for controls)
 * 11) Flick tubing with finger to push air bubbles back into syringe (careful not to flick off syringe)
 * 12) Clamp tubing and then remove the 10-mL syringe
 * 13) Lay flow chambers screw-side-down to prevent air bubbles from entering chamber; lay control chambers screw-side-up
 * 14) Put 4 flows chambers on first and second shelves (2 on each) and 4 control chambers on bottom shelf in incubator
 * 15) Set timer for 30 minutes and let chambers sit in incubator for duration
 * 16) Place PBS on ice or in -20 freezer
 * 17) (For protein) Start thawing lysis buffer and inhibitors

Set-up: Pump
 * 1) Fill a 20-mL syringe with (~15 mL of) media and attach to tubing at the opposite end from the Hamilton syringe
 * 2) Fill tubing with media, bending tubing upwards so that air bubbles keep to the top
 * 3) When media reaches top of the Hamilton syringe, insert plunger down to ~200 μL
 * 4) Remove the 20-mL syringe and insert plastic adapter to end of tubing with the ridged end inserted into the tubing
 * 5) Repeat for 3 additional tubing leads to the pump; test the flow meter to make sure it is connected correctly
 * 6) Cut two pieces of tape
 * 7) Assemble pump by screwing in actuator with tubing attached (be very careful not to pop off tubing from Hamilton syringe)
 * 8) Tape tubing to top of pump with as small an angle as possible
 * 9) Tighten all screws on pump
 * 10) Tape the 1-mL syringes to the wall of the incubator door (2 at a time)
 * 11) Squeeze media through tip of adapter and then attach to tubing on flow chambers
 * 12) Remove clamp and then remove 5-mL syringe from tubing
 * 13) Tighten bolts on Mover 1 of pump
 * 14) While finishing 30-min incubation, set up the 8 other chambers for next experiment

Experiment
 * 1) After 30-min incubation, check all systems and then use Wintest to start experiment
 * 2) Press Run (Zero Start)
 * 3) Record voltages (~1.2 V)
 * 4) Put next set of 8 chambers into incubator and set timer for 30 min
 * 5) Fill two buckets with ice
 * 6) Label 8 Petri dishes (Flow/Control, Cell type) and put ~10 mL of *chilled* PBS into each dish
 * 7) Place Petri dishes on ice in plastic buckets
 * 8) (For Protein Extraction): Prep lysis buffer with protease inhibitors: Add 20 μL of each inhibitor (3 types) to the buffer and store on ice; Vortex mixture (~10 sec)
 * 9) Label microcentrifuge tubes (name, date, cell type, flow/control, sample number); label sample number on top and side of each vial; wear gloves (RNAse free)

Experiment: Removing Chambers wipe cell scraper with EtOH between each sample
 * 1) After 30-min of flow, remove all 8 chambers from incubator
 * 2) Clamp tubing going to pump
 * 3) Remove chamber from adapter
 * 4) Remove screws (using electric screwdriver) and clean in 100% EtOH
 * 5) Slide off top and pull off slide using tweezers
 * 6) Stick slides cell-side-up in PBS in Petri dishes on ice
 * 7) Wash slides 2x with PBS (dump off PBS and pour on a new layer; repeat)
 * 8) Tilt slide on dish top; put cell scraper at bottom of slide (to catch run-off), and then release reagent (800 μL of TriReagent for mRNA or 75 μL of lysis buffer for protein) onto slide
 * 9) Use cell scraper to remove all of cell product from slide and push down into dish
 * 10) Use a P1000 (mRNA) or P200 (protein) to extract all of cell product from dish; Add cell products into microcentrifuge tubes (on ice)
 * 11) Note: If switch between flow and control, use a different cell scraper; switch pipette tips and

Experiment: Cleaning Chambers
 * 1) Hold chamber over a waste beaker and then remove syringe and clamp
 * 2) Dump out media from tubing
 * 3) Remove top of chamber and rinse with 70% EtOH
 * 4) Clean gaskets with 70% EtOH
 * 5) Attach vacuum to tubing and stack to dry

Notes:
 * There are 8 old chambers and 8 new chambers; when upright, new chambers have black hole on left side and old chambers have black hole on right side; new chambers have a purplish tint (with exception of one old chamber)
 * Use new chambers for flow and old chambers for controls
 * Always use a gasket on new chambers (rubber perimeter is higher); one old chamber does not get a gasket and two old chambers have cracked rubber gaskets (use as controls)
 * Don’t have enough screws to use 8 on each chamber; some controls only get 6 screws
 * If tubing does pop off of Hamilton syringe (Step 33), clamp immediately, then use syringe to reapply media throughout tubing (removing clamp); attach tubing to Hamilton syringe (try to keep dry); put a clamp on tubing as assemble pump, as tubing will be more likely to fall off again
 * Lysis buffer and protease inhibitors are located in -20° freezer; RIPA lysis buffer is wrapped in foil and a bag of RIPA lysis buffer kit (inhibitors) are located on bottom shelf
 * TriReagent is located in 4° cold room
 * MLO-Y4 cells plated ~275K cells/slide and TX1/56 plated ~230K cells/slide, 2 days prior
 * Microcentrifuge tubes: For ERK signaling, use 2 slides per data point; for FAK, use 4 slides per data point; For mRNA, use 1 slide per data point

Contact

 * History: J. Litzenberger, last updated 8/9/05

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