Protocols
Cisbio IP-One Tb (62IPAPEC) Assay: 384-well
Microplates
Regular height, 384-well, white, opaque, with lids
Reagent preparation
1x Stimulation Buffer (dilute 5x Stimulation Buffer with water): to resuspend cells and dilute compounds
Cell preparation
Suspension cells: Lift cells with non-enzymatic cell stripper, neutralize with growth medium and count cells. Spin down cells and resuspend in 1x Stimulation Buffer at a desired concentration. (See considerations).
Adherent cells: Lift cells with non-enzymatic cell stripper and seed cells at a desired density 24 hrs prior to the assay (See considerations).
Treatment (28 無 total)
Add 21 無 cell suspension (or 1x Stimulation Buffer for adherent cells) to 384-well white plates + 7 無 4x agonist, incubate 60-90 min at 37蚓.
Adjust reagent volumes when using antagonists.
Termination (40 無 final)
6 無 D2 (IP1-d2, 1:20 in lysis buffer), followed by 6 無 K (Anti-IP1-Cryptate Tb, 1:20 in lysis buffer), incubate 60 min at room temperature, and read on FlexStation 3.
IP1 standards
Prepare during cell treatment, using Stimulation Buffer.
Add 28 μL/well to the plate before D2 and K additions.
| Standard |
Preparation |
Working (μM) |
Final (μM) |
Log[IP1 (M)] |
| Stock |
Reconstituted
reagent in water |
220 |
- |
- |
| A |
5 μL Stock + 95 μL Buffer |
11 |
7.7 |
-5.11351 |
| B |
25 μL A + 75 μL Buffer |
2.75 |
1.925 |
-5.71557 |
| C |
25 μL B + 75 μL Buffer |
0.6875 |
0.48125 |
-6.31763 |
| D |
25 μL C + 75 μL Buffer |
0.171875 |
0.120313 |
-6.91969 |
| E |
25 μL D + 75 μL Buffer |
0.042969 |
0.030078 |
-7.52175 |
| F |
25 μL E + 75 μL Buffer |
0.010742 |
0.00752 |
-8.12381 |
| G |
25 μL F + 75 μL Buffer |
0.002686 |
0.00188 |
-8.72587 |
| H |
75 μL Buffer |
0 |
0 |
-10 |
Considerations
Cell density optimization: test 5-20 K/well.
Be cautious when changing microplate type and/or total reaction volume.
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