Tina:Sample protocols

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Immunostaining protocol[edit]

  • Protocol:

1. Remove PBS from well 1, 2, 3, 4, 5, 7, and 8.

2. Fill well 1 with 350 ul PBS, well 2 with 100 ul 4% paraformaldehyde, well 3 with 100ul blocking buffer (usually 3% BSA + 0.1% TritonX-100 in PBS), well 4 with 50ul primary antibody working solution (volume could be increased for easy dilution; dilute antibody to working solution in blocking buffer(3% BSA + 0.1% TritonX-100)), well 5 with 50ul secondary antibody working solution.

3. Seal plate, type in the script, and run protocol.

4. Take pictures with fluorescence microscope.


  • Script:"
(1)Flow rate used in this script is for H9 ESC, other cell types may need further optimization. 
For example, differentiated cells need slower flow rate.
(2)Antibody hybridization time depends. 
Usually, 60~90 mins for primary antibody, and 30~60 mins for secondary antibody should be enough.

setflow X 0.25

setflow Y 2


% Wash

open V1

wait 10

close V1


% Fix

open V2

wait 20

close V2


setflow X 0.5


% Wash

open V1

wait 15

close V1


setflow X 0.5


% Block and Permeablize

open V3

wait 40

close V3


% Expose to primary antibody solution

open V4

wait 60

close V4


% Expose to secondary antibody solution

open V5

wait 40

close V5


setflow X 0.25


% Wash

open V1

wait 15

close V1


end


Cell culture protocol[edit]

  • Protocol:

1. Remove PBS from well 1, 2, 3, 4, 5, 7, and 8.

Keep Well 1 empty to avoid the interference.

2. Fill well 2, 3, 4, and 5 with 350 ul culture media. (Induction media could be used to induce differentiation at certain point. A gradient induction media is also possible with a combination of X/Y flow rate setting.)

3. Seal plate, type in the script, and run protocol.

4. Acquire bright field stream data with microscope.


  • Script:"
(1)Flow rate used in this script is for H9 ESC, other cell types may need further optimization. 
For example, differentiated cells need slower flow rate.
Sample Script: culture in general culture media for 24 hrs, then changed to osteogenic induction media for 3 days 

setflow X 0.5


% Culture

open V2

wait 1440

close V2


% Induction

open V3

wait 1440

close V3


open V4

wait 1440

close V4


open V5

wait 1440

close V5


end