Fibrin clot formation and external fibrinolysis

See attached for a specific protocol for the turbidity and lysis data presented in the paper. 

We have also attached a more general protocol as PDF file, but the conditions employed in the paper are found on the word document called "Turbidity and external lysis protocol for elife".
 

PROTOCOL: TURBIDITY & LYSIS (FIBRINOGEN)
KEEP ALL SAMPLES AND REAGENTS ON ICE.

Purify Fbg from plasma samples. Quantify [Fbg] using the nanodrop.

PLATE SETTING:

Turn Kinetic Plate Reader and Computer ON, a minimum of 15min before use.

Set up a program to read plate (λ=340nm, every 12s for 120min). Check temperature is at 37°c.

Dilute samples for a final concentration of 0.5mg/ml Fbg, in Permeation Buffer: 240μl per samples (2x 100μl).

Distribute 25μl/well of Fbg standardand samples to 96-well plate (Greiner Bio-One655101), in duplicate (minimum).

TURBIDITY:

Add 75μl/well of Permeation Buffer. Prepare Activation Mix 1.

Place 96-well plate in Kinetic Plate Reader.

Add 50μl/well of Activation Mix 1, with 5 sec gap between each column. Close lid.

Press “Read Plate” (λ=340nm, every 12s for 120-360 min). After 120-360 min, save data.

LYSIS:

Prepare Lysis Mix.

Add 75μl/well of Lysis Mix. Prepare Activation Mix 2.

Place 96-well plate in Kinetic Plate Reader.

Add 50μl/well of Activation Mix 2, with 5 sec gap between each column. Close lid.

Press “Read Plate” (λ=340nm, every 12s for 120-360min). After 120-360 min, save data.

Thrombin: Calbiochem, 605190 (-80°C). Plasminogen: ERL, HPG 1850AL (-80°C). tPA: TechnoClone, TC41072 (-80°C).

rhFbg (standard): Calbiochem xxxxx (-80°C).

Turbidity and Lysis 

KEEP ALL REAGENTS ON ICE

Plate Settings: 

Run at 340 nm, shake at the start 

2-4 hours in length 

Temperature 37°C

Turbidity

1x TBS: 

50 mM Tris (6.06g/l)

100 mM NaCl (5.84 g/l)

pH_7.4 (with 5 M HCl)

Activation Mix:

Remember to make extra for the multichannel

Method

1. Turn on plate reader to heat up to temperature
2. Prepare fibrinogen in TBS at desired concentration with enough for an extra well for pipetting loss. 
3. Add 30 µl of prepared fibrinogen mixture per well into 384 well plate
4. Prepare activation mix (enough for experiment and additional for multichannel).
5. Add 20 µl per well of activation mix (using a multichannel), with 10 sec gap between each column, make sure there are no bubbles. Plate reader reads down a column so add activation mix vertically to the plate.
6. Insert into plate reader and press “read plate” (λ = 340 nm, every 12s for 2 hr at 37 °C) 

External Fibrinolysis 

1. Prepare a lysis mix of 1 nM t-PA and 0.24 µM glu-plasminogen in TBS (15 µL per well, prepare extra for multichannel) 
2. Using a multi-channel pipette gently add the prepared lysis mix on top of the formed clot, add lysis mix in a vertically to wells.
3. Start experiment (read every 12 seconds for 4 hours at 37 °C at 340 nm). 
4. Once finished save and export data