Physiology

Protocol For Platelet Associated Ig Elution

OUTLINE

Platelet-associated Ig (PAIg) could be eluted from the platelets. As a result of this procedure platelets get destroyed and the eluted Ig is formed of platelet surface-bound Ig and , mainly, of internal pools of Ig (from a-granules). That is why the caution must be taken when interpreting results. 

PROTOCOL

I. GLYCINE-HCl Elution

 

  1. Resuspend the platelet pellet in 0,1 mol/L glycine HCl (pH=2.5) to a final concentration 2-10 x 10^8 Platelets/mL
  2. Incubate at RT for 15 min.
  3. Centrifuge at 14000 rpm, 5min , RT or 10??C
  4. Neutralize with 1mol/L Tris-base (pH=9.0)
  5. Dialyze ON, 4??C, against PBS
  6. Centrifuge at 14000 rpm, 5min , RT or 10??C

II. ETHER Elution

 

 

  1. Resuspend the platelet pellet (10^8 Platelets/mL) in 400µl [PBS-EDTA 5mM-BSA 2%] 
  2. add 800µl of diethylether
  3. vigorously mix for 3 minutes 
  4. incubated afterwards at 37??C for 45 minutes in 1,5 ml plastic tube without additional shaking. 
  5. let the tube overnight at 4??C for ether evaporation 
  6. centrifuged at room temperature for 10 minutes at 5000g

SOLUTIONS
I.

 

 

  1. 0,1 mol/L glycine HCl (pH=2.5)
  2. 1mol/L Tris-base (pH=9.0)
  3. PBS

II.

 

 

  1. PBS-EDTA 5mM-BSA 2%
  2. diethylether extra-pure

ADDITIONAL INFO
If the fraction you are interested in is IgM don't freeze the sample. To avoid the contamination in the tube add NaN 3 to the final concentration of 0,05%

 

Protocol For Western Blot With Platelet Proteins

OUTLINE

Western blot is a wide used technique to identify a target protein/s for the certain antibody.

PROTOCOL

  1. Prepare platelets.
  2. Lyse washed platelets (109 platelets/ml) with 1ml of ice-cold complete lysis buffer. Let to stay for 30min on ice.
  3. Remove debris by centrifugation at 100,000 g for 15 min. 
  4. Measure the total protein concentration by a Lowry assay (Lowry et al, 1951).
  5. Add either reducing or non-reducing sample buffer to a protein probe. Mix well. Boil for 5 min.  Centrifuge  for 10 min. at 14  000 rpm (100,000g) (ultracentrifuge) if needed. 
  6. Prepare and load the running gel. Afterwards, prepare and load stacking gel. Use a comb to form loading "wells" for samples of proteins.
  7. Place the gel into the current field (20 mA per stacking gel, 30 mA per running gel)
  8. Transfer proteins to Hybond ECL Nitrocellulose membranes (Amersham Pharmacia Biotech, England) (200 mA-1 A, 2 hours). 
  9. Sink in Ponceau S solution. Get rid of the Ponceau S solution by washing with ddH2O.
  10. Block membranes for 1 hour at room temperature with PBS containing 5% dried nonfat milk.
  11. Wash 1×15 min and 2×5 min in PBS containing 0.05% Tween-20
  12. Incubate overnight at 4??C with the first Ab in PBS containing 5% dried nonfat milk.
  13. Wash 1×15 min and 2×5 min  in PBS containing 0.05% Tween-20
  14. Incubate for 1 hour at room temperature with the right dilution of revealing antibody conjugated to horse-radish peroxidase. 
  15. Wash 1×15 min and 2×5 min  in PBS containing 0.05% Tween-20
  16. Reveal bound antibodies with ECL western blotting detection reagents (Amersham Pharmacia Biotech, England)  according to the manufacturer's instructions. Visualize the chemoluminiscence with the X-Ray film.
SOLUTIONS
  1. complete lysis buffer = 50 mM Tris HCl, 150 mM NaCl, 1mM EDTA, 1% triton X-100, 10% glycerol, 1 mM NaF, 1 µg/ml pepstatin A, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, pH 7.4.
  2. running gel (lower), V=20 ml

    substance/gel conc./ % 5% 7% 10%
    acrylamid/bis-, 40% 2.5 ml 3.52 ml 5 ml
    ddH2O 11.7 ml 10.68 ml 9.2 ml
    lower TRIS 5 ml 5 ml 5 ml
    glycerol 50%, wtare sol. 0.4 ml 0.4 ml 0.4 ml
    Temed 25 µl 25 µl 25 µl
    Ammonium persulfate, 1% 0.8 ml 0.8 ml 0.8 ml
  3. stacking gel (upper), 3%

    substance amount
    acrylamid/bis-, 40% 1.2 ml
    ddH2O 10 ml
    upper TRIS  4 ml
    TEMED 16 µl
    Ammonium persulfate, 1% 0.8 ml
  4. upper TRIS, 500 ml, water sol., pH 6.8 , store at 4??C, use at RT = tris-HCl, 30.3 g (0.5 M) + SDS, 2 g (0.4% final)
  5. lower TRIS, 500 ml, water sol., pH 8.8, store at 4??C, use at RT = tris, 90.85 g (1.5 M) + SDS, 2 g (0.4 % final)
  6. sample buffer, 50 ml, store at -20??C, use at RT

    substance/ conc., times x2 x3
    glycerol 10 ml 15 ml
    SDS 3 g 4.5 g
    upper TRIS 12.5 ml 18.75 ml
    bromphenol blue 10-40 mg 50 mg
    ddH2O up to 50 ml up to 50 ml
    b-mercaptoethanol
    (only for reducing-conditions)
    5 ml 7.5 ml
  7. running buffer (tank), x10, 1 L, water solution, do not correct pH, store at 4??C, use at RT = tris base, 30.3 g (0.25 M) + glycine, 144.1 g (1.92 M) + SDS, 10 g (1% final). 
  8. running buffer x1, 1 L = 100 ml of running buffer x 10 + ddH2O, 900 ml
  9. transfer buffer, x10, 1 L, water solution, pH 8.3 (do not correct pH), store at 4??C, use at RT = tris base, 14.54 g (120 mM) + glycine, 72 g (960 mM)
  10. transfer buffer, x1, 1 L = transfer buffer, x10, 100 ml (10% final)+ methanol (extra), 200 ml (20% final) + ddH2O, 700 ml
  11. Ponceau S, 1 L, keep at RT = Ponceau S, 1g + acetic acid (glacial), 50 ml + ddH2O, up to 1 L
  12. Coomassie dye, 1 L , keep at RT , dark = Coomassie Blue R-250; 1g + C2H5OH (extra), 400 ml + Acetic Acid (glacial), 100 ml + ddH2O, up to 1L
  13. Decoloration solution, keep at RT = CH3OH, 500 ml + Acetic Acid (glacial), 100 ml + ddH2O, up to 1 L
  14. PBS-tween 20 = 2 L of PBS + 1 ml of tween 20.

Protocol For Platelet Preparation

OUTLINE

In order to avoid platelet activation all manipulations must be performed as quickly and as acurate as possible.Work on ice if possible! 

This protocol is based on differential centrifugation that allows to separate platelets from blood cells. Also you can use Robinson procedure (consult the HANDBOOK OF FLOW CYTOMETRY METHODS) 

PROTOCOL

  1. Bleed animals via the plexus retroorbitalis or the tail vein under ether anesthesia
  2. Collect blood into 1,5 eppendorf tubes with ACD (acid-citrate-dextrose; Sigma C-3821) = 1 vol of ACD : 3 vol of blood : 6.6 vol of NaN 3 0,05%-PBS
  3. Cetrifuge at 216-220g , 5-7min , 10??C 
  4. Take & save at 4??C PRP (platelet-rich plasma ) 
  5. Add 1ml of  either NaN3 0,05%-PBS or BSGC to the remainig eythrocytes and other cells
  6. Cetrifuge at 216-220g , 5-7min , 10??C
  7. Take & save at 4??C PRP (platelet-rich plasma )
  8. Add 1ml of either NaN3 0,05%-PBS or BSGC to the remainig eythrocytes and other cells
  9. Cetrifuge at 216-220g , 5-7min , 10??C
  10. Take & save at 4??C PRP (platelet-rich plasma )
  11. Pool saved PRP
  12. Cetrifuge at 1613g , 10min , 10??C
  13. Aspirate supernatant (SN)
  14. Resuspend sedimented platelets in either EDTA 1%-NaCl 0.9% or BSGC
  15. Cetrifuge at 216-220g , 5-7min , 10??C
  16. Resuspend in in either EDTA 1%-NaCl 0.9% or BSGC. Count platelets
  17. Transfer SN into another tube, save at 4??C

SOLUTIONS

  1. ACD = 0.1 mol/L trisodium citrate, 0.11 mol/L dextrose, and 71 mmol/L citric acid monohydrate 
  2. ACD pH 4.9 = trisodium citrate dihydrate 22g/L + citric acid monohydrate 8g/L + dextrose 24.5 g/L
  3. NaN3 0.05%-PBS (for 500ml) = 100ml PBS(x 5) + 400ml ddH2O + 0.25g NaN 3 pH7.0-7.2 
  4. PBS (x5)(phosphate buffer saline x5) (for 5L) = 180g NaCl + 37g Na 2HPO4*2H 2O + 10.75g KH2PO4 + ddHO to 5L
  5. EDTA 1%-NaCl 0.9% (for 500ml)= 5g EDTA + 0.9% NaCl to 500ml pH 7.0 
  6. BSGC = 8.6 mM Na2HPO4, 1.6 mM KH2PO4, 0.12 M NaCl, 0.9 mM EDTA, 13.6 mM Na citrate, 11.1 mM glucose, pH 7.3 (Levin et al, 1999)

ADDITIONAL INFO

  1. it's possible to bleed mice by cardiac puncture under ether anesthesia.
  2. it's possible to collect blood into sodium citrate 3.8% ( 0.1 mol/L ) = 1 vol citrate : 9 vol blood : 50 vol buffered saline-glucose-citrate pH7.3 // or heparin 7.5 U/ml   
  3. RCF = 1.12 x r x (RPM/100) x (RPM/100) where RCF-relative centrifugal force in g  , r -centrifuge radius in mm , RPM-rotations per min in rpm
  4. To avoid contamination of PRP with leukocytes and erythrocytes take PRP-upper&middle layers.

Protocol For Thrombophagocytosis

OUTLINE

The protocol shown below represents an Ab-mediated thrombophagocytosis modification. 

PROTOCOL

I.  Collect platelets. 
II. Label platelets with CMFDA.
III. Coat platelets with an Ab directed to platelet Ag. 
IV. Prepare peritoneal macrophages. 
V. Run Ab-mediated thrombophagocytosis.
 

  1. Add 100µl of peritoneal cells (20×10^6/ml) into 5 ml Polypropylene Round-Bottom tubes (Falcon, Cat.: 35 2063). Add 45 µl of platelet suspension resulted from CMFDA-labeling and Ab-coating step. Vortex at medium speed.
  2. Incubate tubes as duplicates at 37??C and on ice for 60 min.
  3. To stop the phagocytosis the tubes incubated at 37??C should be placed on ice all together at the same time (!).
  4. Add 100µl of ice-cold quenching solution to the each tube. Vortex at the medium speed.
  5. Add 4 ml of ice-cold PBS or washing solution.
  6. Centrifuge at 1200 rpm, at 4??C, for 5 min.
  7. Resuspend in 4 ml of ice-cold PBS or washing solution.
  8. Centrifuge at 1200 rpm, at 4??C, for 5 min.
  9. Resuspend in 4 ml of RT lysing solution. Incubate at RT, for 10 min., at dark.
  10. Centrifuge at 1200 rpm, at 4??C, for 5 min.
  11.  Resuspend in 4 ml of ice-cold PBS or washing solution.
  12. Centrifuge at 1200 rpm, at 4??C, for 5 min.
  13. Resuspend in 200µl of ice-cold DNA-staining solution. Vortex gently.
  14. Run FACS within the next 60 min. 

SOLUTIONS

  1. PBS
  2. washing solution (commercial, comes with the kit)
  3. lysing solution (commercial, comes with the kit)
  4. DNA-staining solution (commercial, comes with the kit)

Serum Separation From Whole Blood

1) Collect sample (preferably in glass tubes) and leave for 1 hour at 37°C to allow it to clot.

2) Leave sample at 4°C overnight to allow the clot to contract.

3) Using a glass pasteur carefully loosen the clot from the sides of the tube. It is important not to lyse the red cells as they can not then be separated from the serum.

4) Centrifuge the serum at 4000 rpm for 20 minutes @ 4°C.

5) Remove the serum from the clot by gently pipetting off into a clean tube using a glass pasteur.

6) Label with the animal number, antigen, total bleed and date.

7) Store at -20°C.

Protocol For Elisa With Platelets

OUTLINE

This modification of qualitative ELISA (Enzyme-Linked Immunosorbent Assay) is used for either screening detection of anti-platelet antibodies or for detection of platelet-associated Ig (PAIg) (shown here).

 PROTOCOL

  1. prepare platelets from control and immunized mice
  2. distribute 2.5×106 platelets per well on F-bottomed MaxiSorp Nunc-Immuno microplates (GibcoBRL, Roskilde, Denmark), centrifuge at 3000 rpm, 7 min, 10??C. 
  3. fix with PBS-PF 2 % for 5 min, RT
  4. wash 3 times in PBS-tween 20, dry
  5. block half of the plate (with platelets) by PBS-BSA 1% and coat another half by glycine (x1) – BSA for 1 hour at 37??C
  6. wash 3 times in PBS-tween 20, dry
  7. incubated at 37??c for 1 hour with a 1:1000 dilution of rat IgG2a anti-mouse kappa chain monoclonal antibody conjugated to horse-radish peroxidase (LO-MK1, LO/IMEX)
  8. reveal with o-phenylenediamine dihydrochloride (OPD) in revelation solution.

SOLUTIONS

  1. PBS-PF (paraformaldehyde), 2%
  2. PBS-tween 20, 0.05%
  3. glycine (x1) – BSA, 1 mg/ml
  4. revelation solution (citrate/phosphate buffer), pH 5, 1 L, water solution = citrique acid, 5.1065 g + Na2HPO4 x 2H20, 9.08 g
  5. glycine buffer (coating solution), pH 9.2, water solution  = glycine 1 M + NaCl 1.5 M 
ADDITIONAL INFO
This protocol may be used for screening of hybridoma supernatants for anti-platelet Ab. In this case, the extra step such as an incubation with a certain SN and additional washing should be added before the step No 7.

Protocols For Coating Of Platelets With An Antibody In Vitro

OUTLINE

Antibody-coated platelets (opsnized) may be used in the subsequent thrombophagocytosis assay. 


PROTOCOL
  1. Resuspend 1.6×10^8 of CMFDA-labeled platelets in 200µl of BSGC-BSA (v/w 1%) either with a control Ab or an Ab directed to platelet Ag (you may use Grener F-bottom microtitration plate for this step). If the Ab used is rat IgG1 (k) against mouse CD41 (MWReg30, Becton Dickinson, Cat.: 09911D) then the advised concentration is 0.6µg/ml (0.15µg per 200µl of suspension). Incubate the suspension for 45 min., at 37??C.
  2. Centrifuge at 3000 rpm at 4??C, for 7 min.
  3. Resuspend in BSGC.
  4. Centrifuge at 3000 rpm at 4??C, for 7 min.
  5. Resuspend in 200µl of IMDM.
  6. Centrifuge at 3000 rpm at 4??C, for 7 min.
  7. Resuspend in 200µl of IMDM-FCS 5% decomplemented.

SOLUTIONS
  1. BSGC
  2. BSGC-BSA 1% (v/w) = 9990ml BSGC+10g BSA
  3. IMDM (Iscove's Modified Dulbecco's Medium)
  4. IMDM-FCS 5% (v/v) heat-decomplemented (56??C, 30min).

Protocol For Cmfda Labelling Of Platelets

OUTLINE

CMFDA (5-chloromethylfluorescein diacetate) is a lipophilic tracer that has an enormous advantage over ordinary tracers (e.g. FITC) because it can be introduced into live (!) cells. Once inside the cell this dye undergo some covalent changes restricting from its passgae to extracellular milieu.

 PROTOCOL

  1. Collect blood and purify platelets.
  2. Resuspend in the certain known volume of BSGC and count platelets. Separate 10×10^6 platelets (negative control). Centrifuge at 3000 rpm at 4??C, for 7 min. Store them resuspended in Iscove's Modified Dulbecco's Medium (IMDM), at 4??C before the FACS preparation starts.
  3. Resuspend 3.2×10^8 platelets in 5 ml of BSGC together with 50µg of CMFDA in 15 ml Falcon Tube. Store for 60 min., at RT, at dark with slight rotation.
  4. Centrifuge at 3000 rpm at 4??C, for 7 min.
  5. Resuspend in 5 ml of BSGC.
  6. Centrifuge at 3000 rpm, at 4??C, for 7 min.
  7. Let to stay for 60 min., at RT, at dark (elimination of CMFDA access)
  8. Centrifuge at 3000 rpm, at 4??C, for 7 min.
  9. Resuspend in the certain known volume of BSGC and count platelets. Separate 10×10^6 platelets (quality control of labeling). Centrifuge at 3000 rpm at 4??C, for 7 min. Store them resuspended in IMDM, at 4??C before the FACS preparation starts.
  10. The main part of the labeled platelets should be centrifuged, resuspended in the respective solution (NaCl 0.9% for in vivo use, IMDM, HCF or PBS for in vitro use), counted, and utilized in subsequent in vitro or in vivo experiments.

SOLUTIONS

  1. BSGC
  2. IMDM (Iscove's Modified Dulbecco's Medium with 25 mM Hepes/with L-Glutamine) BioWhittaker Europe Cat.: BE12-722F

Protocol For C L O T R E T R A C T I O N

OUTLINE
 
Abnormal Clot Retraction indicates on abnormal platelet numbers, abnormal glycoprotein amount or structure, abnormal platelet signaling, abnormal fibrinogen levels, or abnormal fibrinogen structure

PROTOCOL
 

  1. Use either the whole blood or platelet-rich plasma (PRP)
  2. To obtain PRP, collect 500 µl of blood on 50 µl of ACD
  3. Add 600 µl of PBS, centrifuge once at 10??C, 1200 rpm ( Joan, GR 412)
  4. In Glass Centrifuge Tube add 100 µl of PRP and 160µl of clot retraction buffer, and 50µl of  working solution of thrombin (8 U/mL) and 5-10 µL of erythrocytes (for visualization). Vortex briefly.
  5. Place the Dead End Glass Rod (use Pasteur Pipette)  to the bottom of the tube. Place the tube at 37??C. Check the CLOT FORMATION (appearance) and RETRACTION. Measure the volume of remained solution after clot retraction, notice the color of solution, take digital picture.

SOLUTIONS
 
  1. Clot Retraction Buffer: 400 mL of PBS x1 + 10 mL of PBS-MgCl2 (MgCm2=4g/L) + 10 mL PBS-CaCl2 (CaCl2= 5.3 g/L)
  2. Working Solution of Thrombin = 7 µl of Thrombin (1250U/mL) + 1 mL of Clot Retraction Buffer
 

ADDITIONAL INFO

The Clot Retraction Test may be used for screening of new anti-platelet drugs, anti-platelet-antibodies, or unexplained bleeding-problems. This test does not indicate exactly what is the problem but shows the direction to look for it.

Bleeding Time Test

OUTLINE

Although in the Review Article : "Idiopathic Thrombocytopenic Purpura: A practice Guideline Developed by Explicit Methods for the American Society of Hematology" bleeding time is considered as unnecessary/inapropriate to establish the diagnosis of ITP it's still used in experimental models of ITP study as an additional test.

PROTOCOL

  1. Anesthesize a mouse
  2. Make a standard incision on the dorsal tail vein using Simplate blades
  3. Immerse the tail in NaCl 0.9% at 37.5??C
  4. Record the time from the moment blood is observed to emmerge from the wound until cessation of blood flow

ADDITIONAL INFO

Alternatively you can perform a standard incision with an automatic device (Surgicutt; ITC, Edison, NJ) and every 15 secs apply a filter paper (Whatman 3MM) for 5 sec until the blood no longer stained it.