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):
   1. 4 long pieces (2 cabinet lengths)
   2. 4 medium pieces (6 in)
   3. 32 short pieces (1 decimeter)
4. Attach two short pieces to each chamber
5. Break 4x 1-mL pipettes and discard tapered end; remove cotton with tweezers and then use

pipettes to attach long tubing to medium tubing

1. Attach other end of long tubing to Hamilton syringes on pump
2. Open Wintest
   1. Mover 1/2  Local  Switch from Off to High
   2. Mover 1/2  Waveform  900 cycles = 15 min (7200 c = 2 hr); Amp ± 4.2 mm
3. Put padding (3 large pieces) in incubator
4. Insert flow meter into tubing
5. Pour media into ~6 Falcon tubes
6. Remove locking mechanism from 8x 5-mL syringes and 2x 10-mL syringes and fill with media (from 50-mL conical tubes)
7. 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

1. Remove any air bubbles from media using a bulb
2. Place slides cell-side-down on top of chamber
3. Check that slide is in place using tweezers
4. Remove excess media with bulb
5. Put gasket down on top of slide (cover black rubber perimeter)
6. 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
7. Tap chamber on bench (tubing upward) to remove air bubbles (don’t need to do for controls)
8. Flick tubing with finger to push air bubbles back into syringe (careful not to flick off syringe)
9. Clamp tubing and then remove the 10-mL syringe
10. Lay flow chambers screw-side-down to prevent air bubbles from entering chamber; lay control chambers screw-side-up
11. Put 4 flows chambers on first and second shelves (2 on each) and 4 control chambers on

bottom shelf in incubator

1. Set timer for 30 minutes and let chambers sit in incubator for duration
2. Place PBS on ice or in -20 freezer
3. (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

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

1. After 30-min of flow, remove all 8 chambers from incubator
   1. Clamp tubing going to pump
   2. Remove chamber from adapter
2. Remove screws (using electric screwdriver) and clean in 100% EtOH
3. Slide off top and pull off slide using tweezers
4. Stick slides cell-side-up in PBS in Petri dishes on ice
5. Wash slides 2x with PBS (dump off PBS and pour on a new layer; repeat)
6. 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
7. Use cell scraper to remove all of cell product from slide and push down into dish
8. Use a P1000 (mRNA) or P200 (protein) to extract all of cell product from dish; Add cell products into microcentrifuge tubes (on ice)
9. Note: If switch between flow and control, use a different cell scraper; switch pipette tips and

wipe cell scraper with EtOH between each sample

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

or instead, discuss this protocol.