Tina:Sample protocols
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