Video Manual

Basic Usage
  • How to add liquid
  • How to discard liquid
  • How to move SIEVWELL Slide
Protocol
  • Pre-wetting
  • Replace assay medium
  • Cell loading
  • Cell Culture
  • Fixing, Permeabilization
  • Blocking
  • Staining with a typical staining solution
  • Staining with minimum amount of staining solution
  • Observation

Basic Usage

How to add liquid

  1. Hold both ends of the body tightly during liquid handling.
  2. Liquid should be gently flown into the chamber along with the frame wall by touching pipette tip at the corner of the frame.
  3. Tilt the pipette tip against the device during liquid handling to prevent breaking the membrane. Touching pipette tip to the membrane might break the membrane.
  4. Load liquid gently and slowly (ca. 5 seconds for 1 mL loading). Gentle liquid loading is important to keep the trapped cells within the nanowells and to obtain a high single cell rate.

How to discard liquid

Using multi channel pipette version

  1. Set an 8-channel multi-channel pipette. Attach tips to the 1st and 5th pipette channels.
  2. Tightly put the pipette tips to the holes in two side ports, then aspirate liquid.
  3. Aspirate proper amounts of liquid slowly with careful attention to level of the liquid surface (ca. 3 seconds for 200 µL x 2 ports aspirating

Using single pipette version

  1. Completely close one of side ports with a P200 tip and finger.
  2. Tightly put a pipette tip into a hole in the opposite side port.
  3. Aspirate slowly and carefully by watching the level of the liquid surface.

Do not let the filter membrane dry up. If liquid disappears above the membrane, stop aspirating. After aspiration, thin layer of liquid remains on the surface of the membrane. Do not aspirate too much liquid. Once film is dried up, liquid is difficult to penetrate through the membrane and air bubble comes under membrane.

How to move SIEVEWELL Slide

Please hold the protruding part to move the SIEVEWELL Slide.

Protocol

Pre-wetting

    1. Add 1 mL of 100 % ethanol to the chamber.
    2. Wait until ethanol fully wet the membrane.
    3. Immediately add 1 mL of distilled water to the chamber.
    4. Discard 990 μL from side ports. (165 µL x 2 ports x 3 times).
    5. Add 2 mL of distilled water to the chamber.
    6. Discard 2 mL from side ports. (200 µL x 2 ports x 5 times).
    7. Repeat steps 5 and 6 for 2 times.

Replace assay medium

    1. Add 2 mL of assay medium to the chamber.
    2.  Discard 2mL from side ports. (200 µL x 2 ports x 5 times).
    3. Repeat steps 1 and 2 for 2 times

Cell loading

Recommended number of cells: 

  ○ SWS2001-5: less than 2×10^5cells/mL

  ○ SWS5001-5: less than 5×10^4cells/mL

    1. load 1 mL of cell suspension.
    2. Discard 1 mL from side ports. (125 µL x 2 ports x 4 times).
    3. Go immediately to next step. Do not leave the device more than 5 min at this step.

Cell Culture

Add medium before incubation

    1. Add 1mL of assay medium to the chamber.
    2. Cover with lid to avoid drying up surface.

When you replace culture medium…

  1. Discard 1 mL from side ports. (125 μL x 2 ports x 4 times)
  2. Add 2 mL of assay medium to the chamber.
  3. Discard 2 mL from side ports. (200 µL x 2 ports x 5 times).
  4. Repeat steps 2 and 3 for 2 times to replace culture medium completely.

Fixing, Permeabilization

Acetone cannot be used for fixation reagnet in SIEVEWELL.

    1. Add 500 µL of fixation reagent to the chamber.
    2. Discard 100 μL from side ports. Fixing reagent penetrate into nanowells by this operation.(50 µL x 2 ports x 1 time)
    3. Incubate for 15 minutes at room temperature (for 4% PFA, incubation time will vary depending on type of fixation reagent). Cover the chamber with the lid.
    4. Discard 400 µL from side ports. (200 µL x 2 ports x 1 time)
    5. Add 2 mL of PBS to the chamber.
    6. Discard 2 mL from side ports. (200 µL x 2 ports x 5 times)
    7. Repeat steps 5 and 6 for 2 times.
    8. Add 500 µL of permeabilization reagent to the chamber.
    9. Discard 100 μL from side ports. (50 µL x 2 ports x 1 time)
    10. Incubate for 10 minutes at room temperature (for 0.2% TritonX-100/PBS, incubation time will vary depending on type of fixation reagent). Cover the chamber with the lid.
    11. Discard 400 µL from side ports. (200 µL x 2 ports x 1 time)
    12. Add 2 mL of PBS to the chamber.
    13. Discard 2 mL from side ports. (200 µL x 2 ports x 5 times)
    14. Repeat steps 12 and 13 for 2 times.

Blocking

    1. Add 1 mL of blocking reagent to the chamber.
    2. Discard 200 μL from side ports.(100 µL x 2 ports x 1time)
    3. Incubate for 15 minutes at room temperature (for Protein block, incubation time will vary depending on type of fixation reagent). Cover the chamber with the lid.
    4. Discard 800 μL from side ports.(200 μL x 2 ports x 2times)

Staining with a typical staining solution

    1. Add 1 mL of staining reagent to the chamber.
    2. Discard 200 μL from side ports.(100 µL x 2 ports x 1time)
    3. Incubate by following time and temperature recommended for your staining solution. Cover the chamber with the lid to avoid drying up the membrane.
    4. For incubation over 1 h, recommend to incubate within
      humidified chamber to prevent drying up the membrane.
    5. Discard 800 μL from side ports.(200 μL x 2 ports x 2times)
    6. Add 2 mL of PBS to the chamber.
    7. Discard 2 mL from side ports. (200 µL x 2 ports x 5 times)
    8. Repeat steps 6 and 7 for 2 times.
    9. Add 1 mL of PBS to the chamber.

Staining with minimum amount of staining solution

  1. Add 2 mL of 0.01% Tween20/PBS (=staining buffer) to the chamber.
  2. Discard 2 mL from side ports.(200 µL x 2 ports x 1 time)
  3. Carefully discard all the residual liquid at the four corners by aspirating 20 µL from side ports (10 µL x 2 ports) for several times.
  4. Add 300 μL of staining reagent to the chamber.
  5. Incubate by following time and temperature recommended for your staining solution. Cover the
    chamber with the lid to avoid drying up the membrane. For incubation over 1 h, recommend to incubate within humidified chamber to prevent drying up the membrane.

Observation

Set SIEVEWELL Slide on your microscope stage as for a slide glass.

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