TITLE: Molding microchambers in agar with PDMS stamps for live imaging
AUTHORS: Tara Essock-Burns, Galo Garcia, David Matus

[DESCRIPTION]
This is a step-by-step guide for preparing agar microchambers using a patterned stamp. The protocol includes detailed instructions on selecting the appropriate stamp, preparing the setup, centrifuging the agar into the stamp, and mounting cells of interest for live imaging.

[STEPS]
SECTION: Gather materials and prepare setup
1. Familiarize yourself with the protocol and make sure to click the appropriate tab based on whether this is your first time doing this, a subsequent time, or your first time using a new stamp.
SECTION: Gather materials and prepare setup
5. Stack four glass slides and bind each end of the stack with a single layer of lab tape. You will need two bound stacks of four to make one agar pad. If you are making two agar pads at once, you will need four slide stacks total.

  
 

SECTION: Gather materials and prepare setup
2. Review the workflow below. We refer back to this diagram a few times throughout the protocol.
   
SECTION: Gather materials and prepare setup
3. Purchase a stamp from researchmicrostamps.com (or equivalent) with protrusions in the desired dimensions. See Figure 9 in our main publication to estimate the appropriate size.  

Once you have the stamp, make sure you can tell which side has the protrusions.
SECTION: Gather materials and prepare setup
4. Wash the stamp with 70% ethanol in deionized (DI) water and allow to air-dry completely. You can dry in a one-well plate to minimize dust. If small fibers or dust remain on the stamp, try using an air blower/duster.


 

SECTION: Gather materials and prepare setup
2. Wash the stamp with 70% ethanol in deionized (DI) water and allow to air-dry completely. You can dry in a one-well plate to minimize dust. If small fibers or dust remain on the stamp, try using an air blower/duster.
SECTION: Prepare slide sandwich
8. Place a single glass slide on a flat surface lined by each stack of slides (from step 5). This will become the "stamp slide" coded in teal in panel B in our overview figure from step 2.

   
 

SECTION: Melt the agar aliquot
3. Use a heat block set to 100 °C  to melt an aliquot of agar and then transfer to another heat lock set to 70 °C to maintain as a liquid and ensure that no bubbles are in the agar. 
  

SECTION: Prepare the agar
6. Make up 5% agar in the appropriate medium (for more detail see 6.1).
 
 
 

SECTION: Prepare the agar
6.1. To make up 5% agar in a solution, first tare a balance with the weigh boat or paper you will use. Then measure 2.5 g of Noble agar and transfer to a glass container (large enough to hold the volume needed with room for the agar to boil and short enough to fit into the microwave). Add 50 mL of water or medium to the jar. Microwave in 30 s intervals until you see bubbling. Gently swirl the bottle to make sure the agar is fully dissolved. Be careful not to overheat and spill the solution.

In the photo in step 6, we show 50 mL of agar in a 250 mL glass jar.

 

SECTION: Gather materials and prepare setup
2. Follow the "First time" protocol through step 7 and then proceed with the following steps for centrifugation. 
SECTION: Gather materials and prepare setup
4. Pour warm agar over the stamp, covering it completely within the well. Then cover the plate and quickly transfer the plate to the centrifuge.

  
 


SECTION: Remove stamp and trim agar
13. Remove the slide stacks from both sides of the perpendicular slide sandwich.

  
SECTION: Prepare the agar
7. Place the melted agar into a bead bath or water bath set to 80 °C for 3 min or until the bubbles dissipate. 

SECTION: Prepare slide sandwich
9. Pour the liquified agar on top of the stamp. ~2 mL should be sufficient to cover it.
SECTION: Prepare slide sandwich
10. Place a clean slide perpendicularly on top of the agar.

  
SECTION: Prepare slide sandwich
11. Place a metal object on top of the agar+slide sandwich to apply even pressure for at least 1 min or up to 5 min.

  
SECTION: Prepare slide sandwich
12. Transfer the slide sandwich to cool at 4 °C for at least 30 min or up to 60 min. You can move the entire setup to the lid of a well plate.

  
SECTION: Remove stamp and trim agar
14. Use a razor blade to gently pry the bottom slide off of the sandwich.

  
 

SECTION: Remove stamp and trim agar
15. Use the razor blade to score (cut through the top, about halfway down) all four sides of agar along the edges of the stamp.

  
SECTION: Remove stamp and trim agar
16. Once the agar is scored around all edges, you should be able to use forceps to gently peel off and discard or recycle the thin layer of agar on top of the stamp.
SECTION: Remove stamp and trim agar
17. Use the forceps to gently dislodge the stamp without piercing the agar microchamber pad.

  
SECTION: Remove stamp and trim agar
18. Trim the walls of the agar pad using the razor blade to leave a uniform square.

  
 

SECTION: Mount samples for imaging
19. Use a microscope to check that you have clean, intact agar microchambers before mounting.

    
SECTION: Add agar to the slide setup
4. Pour ~2 mL of liquid agar onto the stamp in the slide setup. Apply even pressure with a metal object and transfer the setup on a lid of a plate to 4 °C for at least 30 min.
  
SECTION: Add agar to the slide setup
5. Gently disassemble slide setup, remove the stamp, and trim excess agarose. Cut to divide the agar microchamber into four squares for four different treatments, if desired.
SECTION: Mount samples for imaging
21. Prepare your cells or organisms of interest at the desired density. 

 

SECTION: Mount samples for imaging
22. Pipette 2–5 µL of cells or organisms onto the agar pad, spreading the liquid across to cover multiple microchambers.

  
 

SECTION: Mount samples for imaging
20. Melt an aliquot of VALAP at 80 °C using a heat block. It should be liquid after 5 min.

 
 
 
SECTION: Mount samples for imaging
23. Once the ratio of cells/organisms in the wells is satisfactory, place a clean, square coverslip (#1.5 thickness) and seal with VALAP. 

 

SECTION: Mount samples for imaging
24. Mount the slide onto the microscope stage and begin imaging.

  
SECTION: Load sample
6. Pipette cells or organisms at the desired concentration onto the agar microchamber and seal with a coverslip if using an immersion objective.
SECTION: Gather materials and prepare setup
3. Place with the protrusions facing up into one well of a six-well plate. 

  
 


SECTION: Gather materials and prepare setup
5. Spin the agar into the stamp 700 x g, 3 min, 37 °C.

  
SECTION: Move agar+stamp to slide setup
6. Use forceps or a metal scoop to lift the stamp with agar from the plate and place onto the slide setup.
SECTION: Move agar+stamp to slide setup
7. Resume the protocol at step 11 in the "First time" version.