TITLE: Protocol for micro-sheet pocket 1: Detailed operation manual of six requirements
AUTHORS: Keiichi  Iwaya, H-Arai  , Nanao  Takatou, Yuka Morita, Rinko  Ozeki, Hirofumi  Nakaoka, Masaru Sakamoto, Tsutomu  Kouno, Masayoshi  Soma

[DESCRIPTION]
This protocol details micro-sheet pocket 1 and detailed operation manual of six requirements.

[GUIDELINES]

Test environment: A horizontal worktable in a laboratory in accordance with JIS 8703-1983, ISO 554-1976 (temperature 23°C, relative humidity 50%) 

[STEPS]
SECTION: Average diameter of filter opening
1. Examine the average diameter of filter opening by referring to the catalog of the sheet.
SECTION: Average diameter of filter opening
3. Confirm opening size by using scanning electron microscopy (SEM), if possible.

 
SECTION: Chemical resistance
9. 
Cut in half the sheet in order to compare each other.
SECTION: Water permeability test
14.  Make-up for water permeability test device

SECTION: Water permeability test
15. Create a sample reservoir
SECTION: Water permeability test
15.1. Cut the attachment part (a) of the tip of the P1000 pipette with scissors from the tip body (b) indicated by red arrow. 


  

SECTION: Water permeability test
15.2. For one sheet, cut off at 2 cm from the tip of (b). In the case of two or more sheets, cut off 1.5 cm from the tip. (Red line).


  

SECTION: Water permeability test
15.3. Measure the inner diameter (cm) of the cut side of (b) and calculate the cross-sectional area (inner diameter x inner diameter x 3.14) (Cross-sectional area of test sheet). 
SECTION: Water permeability test
15.4.  Cut a 2 cm area from the opening of the tip of the P200 pipette with scissors (c). 
SECTION: Water permeability test
15.5. Cut a 3 cm area from the opening of the other P200 pipette tip with scissors (d).
SECTION: Water permeability test
15.6. Connect (b) Connect (c) and (d) (yellow arrow). 
SECTION: Water permeability test
15.7. Cover (b) with the test sheet (3cm×3cm) on the red line. 
SECTION: Water permeability test
15.8. Fix the sheet by fitting (a) (blue arrow).
SECTION: Water permeability test
15.9. Cut the sheet that protrudes from (a). 
SECTION: Water permeability test
15.10. Seal and fix the connected parts with Parafilm.
 

 
  



   

 

SECTION: Water permeability test
16. Vertical installation of a water storage tank on the workbench using a Burette stand. 
SECTION: Water permeability test
17. Assemble water storage tank, drainage tank 1, drainage tank 2, according to the reference above diagram. 
SECTION: Water permeability test
18. Adjust the height of the overflow opening to "5 mL" on the scale of a 50 mL disposable measuring pipette.

SECTION: Water permeability test
19. Attach the sample reservoir to the end of the reservoir.
SECTION: Water permeability test
20. Wrap the parafilm to prevent liquid leakage. 
SECTION: Water retention test
23. Prepare 3 sheets cut in 100 mm x 100 mm sized square. 
SECTION: Water retention test
24. Measure the mass of a cut sheet in the standard condition to 1 mg-level. 

SECTION: Water retention test
25. Immerse 3 sheets in the water for at least 15 min. 

SECTION: Water retention test
26. Remove 3 sheets from the water with tweezers. 
SECTION: Water retention test
27. Allow the water to drip off for at least  1 min

SECTION: Water retention test
28. Measure the mass of a cut sheet after wetting and dripping off the water (m2). 
SECTION: Water retention test
29. Calculate the water retention rate by the following formula 


                                   m=(m2-m1)/m1 x 100 

      
                           m: water retention rate (%) 

                           m1: mass of the specimen in the standard condition (mg) 

                          m2: mass of the specimen after wetting and dripping off the water (mg) 




SECTION: Cell transit test
31. Preparation of sample reservoir

Preparation of sample reservoir (consisting of part (a) and (b), we named“filter assembly”) is explained in step 15.1
SECTION: Average diameter of filter opening
2. Observe under a polarized light microscope for sheets with a diameter of filter opening of less than 10 μm.

SECTION: Thickness
4. Examine the thickness by referring to the catalog of the sheet.
SECTION: Thickness
8. After about 10 s , measure the test pieces to 0.1 mm using a vertical ruler.

SECTION: Thickness
7. Lay a glass plate (200 ± 2 mm square, a mass of 82 ± 2 g, a thickness of    ) on the test sheets.

SECTION: Thickness
6. Put 10 test sheets on a horizontal worktable.
SECTION: Thickness
5. Prepare 10 test sheets (cut with scissors to 20 cm x 20 cm square ).

SECTION: Chemical resistance
13. If no obvious change is observed, repeat the procedure 9-12 by changing the test solution (formalin, methanol).
SECTION: Chemical resistance
12. Wash with water, dried, and observe both samples.
SECTION: Chemical resistance
11.  Cover the sheet with a lid and kept at   Room temperature  for about 480 min .

SECTION: Chemical resistance
10. Immerse the whole part of one cut sheet in the test solution (xylene).
SECTION: Water retention test
30. Calculate the average value in accordance with JIS A 5209:2014, Rule B (rounding method). 
SECTION: Water permeability test
14.1. Create a water storage tank

Cut off the suction opening of the 50 mL disposable measuring pipette with a plastic cutter.

SECTION: Water permeability test
14.2. Create drainage tank 1

Drill a hole to make an overflow port at a height of about 12 cm from the bottom of the container 1 (inner diameter of hole, about 15 mm) .

SECTION: Water permeability test
21. Operation Procedure
SECTION: Water permeability test
21.1. Fill drainage tank 1 with water until it flows out of the overflow port.
SECTION: Water permeability test
21.5. Measure t2-t1 value 5 times.
SECTION: Water permeability test
21.4. Repeat the operation in steps 21.1 and 21.2 and confirm that the t2-t1 value was almost constant.
SECTION: Water permeability test
21.3. Measure transit time (t2-t1 value) it takes for the water in a water storage tank to pass from the "40 mL" scale to the "20 mL" scale using a stopwatch.
SECTION: Water permeability test
22. Calculation

The permeability coefficient was calculated by the following formula, applying JIS A 1218:2020.
  

SECTION: Cell transit test
33. Preparation of cell suspension (collection of cells from ascites and/or pleural fluid)


SECTION: Cell transit test
33.1. Centrifugation residual specimens (ascites and/or pleural effusion)at 2000 rpm for 5 min.
SECTION: Cell transit test
33.5. Stand still for 15 min.
SECTION: Cell transit test
33.4.  Add 30 mL to 35 mL of PBS and mix by inverting. 
SECTION: Cell transit test
33.3. Collect more than 1 mL of cell pellet. 
SECTION: Cell transit test
33.2. Gently remove the supernatant with an aspirator. 
SECTION: Cell transit test
33.7. Repeat PBS wash (step 32.4 - 32.6 ).
SECTION: Cell transit test
33.6. Gently remove the supernatant with an aspirator.
SECTION: Cell transit test
33.8. Counts all cells in a cell suspension
Add 90 µL of 0.4% trypan blue solution to 10 µL of cell suspension (10-fold dilution). 
Gentle vortex for 2 s-3 s.
Inject 6 µL samples with a P20 pipette through the sample inlet of the cell counter plate. 
Count the number of cells in 4 compartments using the cell counter plate. 
Calculate the number of cells per 1 µL of stock solution {(average number of cells of 4 compartments) × 10 × (dilution factor)}.

 

SECTION: Cell transit test
34. Adjust to 1 x 10 6- 10 7 cells/ml in 10% neutral buffered formalin solution.
SECTION: Cell transit test
35. Operation Procedure

SECTION: Cell transit test
35.1. Apply 600 µL of cell suspension (1 x 106-107  cells/ml) to the sample reservoir (filter assembly) with a P1000 pipette. 

SECTION: Cell transit test
35.3. Measure the number of cells in the filtrate
Write an enclosure on a glass slide with an alcohol-resistant marking pen.
Add 15 µL drops of 95% ethanol in the enclosure, followed promptly by 15 µL  drops of filtrate.
Allow to natural drying.
Perform Papanicolaou staining.
Count cells in 10 fields of view at 400x under the microscopy.
SECTION: Cell transit test
35.2. Allow to natural filtration. 
SECTION: Water permeability test
21.2. Fill water storage tank with water by using a funnel.
SECTION: Cell transit test
32. Set sample reservoir onto 2 mL microtube.