Oct 29, 2025

Public workspaceCalcium test: preparations, imaging

DOI
https://dx.doi.org/10.17504/protocols.io.ewov11m8pvr2/v1
  • Sofia Galkina1,2,
  • Fedor A. Balabin1,
  • Anastasia N. Sveshnikova1,2
  • 1Center for Theoretical Problems of Physico-Сhemical Pharmacology, Russian Academy of Sciences, Moscow, Russia;
  • 2National Medical Research Centеr of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, Moscow, Russia
Sofia V. Galkina
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov11m8pvr2/v1
Protocol Citation: Sofia Galkina, Fedor A. Balabin, Anastasia N. Sveshnikova 2025. Calcium test: preparations, imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11m8pvr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: October 15, 2025
Last Modified: October 29, 2025
Protocol Integer ID: 229893
Keywords: calcium response of single platelet, calcium test, immobilized platelet, single platelet, using tirf microscopy, tirf microscopy, calcium response, calcium, preparation of whole blood, adhesive coating, types of adhesive coating, assembly of flow chamber, flow chamber
Funders Acknowledgements:
Russian Science Foundation
Grant ID: 23-45-10039
Abstract
This protocol describes a method for recording the calcium response of single platelets in surface-immobilized platelets with a calcium-sensitive dye in a plane-parallel flow chamber using TIRF microscopy.

This includes: preparation of two types of adhesive coatings in flow chambers, collection and preparation of whole blood for the experiment, design and execution of the experiment.

Note: silanization of coverslips and assembly of flow chambers are described in a separate protocol.
Materials
  1. Human thrombin (Hematologic Technologies, Essex Junction, VT, USA);
  2. Non-fibrillar human collagen type I (IMTEK, Moscow, Russia);
  3. CalBryte-590 AM (AAT Bioquest, Sunnyvale, CA, USA);
  4. VM64 antibodies against PECAM-1/CD31 (RRID:AB_782149) was a kind gift from Prof. A. V. Mazurov [3].

All other reagents were from Sigma‐Aldrich (San Diego, CA, USA) unless otherwise indicated.

5. ADP
6. Pluronic F-127
Troubleshooting
Before start
A typical experiment consists of two runs with different flow chambers: one with a collagen coating and one with anti-CD31 coating. The steps for each chamber are identical.

Experiments were performed with whole blood of healthy donors and patients. Whole blood was collected into 1.6 ml hirudin-containing tubes (S-Monovette®, SARSTEDT AG & Co. KG, Nümbrecht, Germany). Samples were stored in a water bath at 37°C until the start of the experiment.

This protocol involves two buffer solutions:

1. Tyrode’s buffer (as in [1])
137 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.36 mM NaH2PO4, 1 mM MgCl2, 2mM CaCl2, 5 mM HEPES (pH 7.5), 0.36% BSA, 1 g/l D-glucose, pH 7.35

2. BRB-80 buffer (as in [2])
80 mM PIPES/KOH, pH 6.9, 1 mM EGTA, 1 mM MgCl2
Preparation of collagen coating
46m 30s
Collagen type I coating simulates physiological conditions near the site of blood vessel damage.

Prepare the following solutions and items:

  1. 100 μl of 1 mg/ml human collagen type I diluted in 0.01M acetic acid (A)
  2. 100 μl of 5% BSA solution in Tyrode’s buffer (B)
  3. 100 μl of Tyrode’s buffer (C)
  4. 1 ml syringe
  5. Empty flow chamber
15m
Connect the syringe to the output tube of the flow chamber and place the input tube into the tube with solution A.
30s
Drive in the solution by pulling the syringe plug and wait for 15 minutes. Always leave a small amount of solution in the tube to prevent air from entering the flow chamber.
15m
Replace solution A tube with solution B tube. Drive in the solution and wait for 15 minutes.
15m
Replace solution B tube with solution C tube. Drive the solution in. Now the collagen-coated flow chamber is ready for use.
1m
Preparation of antibody coating
39m 30s
Anti-CD31 (VM64) coating simulates conditions for low-activated platelets in the periphery of the thrombus.

Prepare the following solutions and items:

  1. 3 x 100 μl BRB-80 buffer (W1, W2, W3)
  2. 100 μl of 40 μg/ml of anti-CD31 (VM64) dissolved in BRB-80 (A)
  3. 100 μl of 1% Pluronic F-127 dissolved in BRB-80 (P)
  4. 100 μl of Tyrode’s buffer (T)
  5. 1 ml syringe
  6. Empty flow chamber
15m
Connect the syringe to the output tube of the flow chamber and place the input tube into the tube with 100 μl of solution W1. Drive in the W1 solution.
30s
Replace solution W1 tube with solution A tube. Drive in the solution and wait for 15 minutes.
15m
Replace solution A tube with solution W2 tube. Drive in the solution. 
30s
Replace solution W2 tube with solution P tube. Drive the solution in and wait for 7 minutes. 
7m
Replace solution P tube with solution W3 tube. Drive in the solution. 
30s
Replace the solution W3 tube with the solution T tube. Drive in the solution. Now the VM64-coated flow chamber is ready for use.
1m
Platelet fluorescent labeling
30m
Load 300 μl of whole hirudinated blood with 2 μM of CalBryte-590 AM and 0.1 u/ml of apyrase, incubate in a water bath set at 37°C for 30 minutes. Use immediately.
30m
Materials and equipment for imaging
  1. Whole blood loaded with CalBryte-590 (as per previous step)
  2. Flow chambers loaded with VM64 and collagen type I
  3. Syringe pump SPLab1, Shenchen
  4. 1 ml syringe with a needle that fits tightly into a silicone tube with an inner diameter of 0.5 mm;
  5. A fluorescence microscope equipped with a video camera (*)
  6. Three 200 μl portions of Tyrode’s buffer
  7. 200 μl of 10 μM ADP solution in Tyrode’s buffer
  8. 200 μl of 10 μM ADP and 5 nM thrombin solution in Tyrode’s buffer.

(*) we used Nikon Eclipse Ti-E with CFI Apochromat TIRF 100XC Oil objective, Nikon LU-N4 laser excitation source, Nikon TI-TIRF-E TIRF module, Andor DU-897 digital EMCCD camera. To record the position and shape of platelets, the DIC mode was used, and to record the fluorescent signal, low-angle TIRF mode with an angle of 48 degrees was used. The power of the 561 nm laser was 5% of the maximum. Frames were collected at 12.25 FPS with 80 ms exposure and no delay.
Flow chamber imaging
1h 5m
Place the first flow chamber on the microscope table and adjust the microscope settings.
7m
Put one tube of a flow chamber into the tube containing blood loaded with a fluorscent due and attach the other to the syringe pump. Use an extension tube when necessary. Set the pump at 33 μl/min rate.

In most cases, such a rate is enough to wash away unbound cells in several minutes.
3m
Wash in nearly 150 μl of whole blood loaded with fluorophore. Observe how platelets are retained and adhere to the coating within the field of view. Wait until approximately 10-15 single cells adhere.

Note: If cells do not adhere well, reducing the speed to 7-15 μl/min may help.
5m
Wash in 150 μl of Tyrode’s buffer, then replace the tube and rinse the chamber with fresh 150 µl of buffer to remove the free-flowing platelets. If the chamber is not sufficiently clear of unattached cells, wash in another 150µl. Set the pump at 33 μl/min rate until the end of the experiment.

Note: the tube needs to be replaced because of blood remains in the first one.
5m
Select a field of view with 10-30 platelets that do not form thrombi or overlap with each other. Take a picture of this field in DIC mode, replace the tube with buffer. Start a recording a 5-minute fluorescence video simultaneously with starting the pump. The buffer must be pumped through the chamber during the entire recording period.

This step allows to measure the level of platelet activation without adding an activator, the base activation level.
5m
Change the field of view and take a picture in DIC mode. Replace the tube for 200 μl of 10 μM ADP solution in Tyrode’s buffer, record fluorescent signal for 5 minutes while pump is working. The activator usually arrives in the middle of the chamber in 30-50 seconds after the pump is turned on.
5m
Change the field of view and take a picture in DIC mode. Replace the tube for 200 μl of 10 μM ADP and 5 nM thrombin solution in Tyrode’s buffer, record fluorescent signal for 5 minutes while pump is working.
5m
Repeat all steps for the second flow chamber.
30m
Protocol references
1) Ignatova, A. A., Suntsova, E. V., Pshonkin, A. V., Martyanov, A. A., Ponomarenko, E. A., Polokhov, D. M., ... & Panteleev, M. A. (2021). Platelet function and bleeding at different phases of childhood immune thrombocytopenia. Scientific Reports, 11(1), 9401. doi: 10.1038/s41598-021-88900-6

2) Gudimchuk, N., Tarasovetc, E. V., Mustyatsa, V., Drobyshev, A. L., Vitre, B., Cleveland, D. W., ... & Grishchuk, E. L. (2018). Probing mitotic CENP-E kinesin with the tethered cargo motion assay and laser tweezers. Biophysical journal, 114(11), 2640-2652. doi: 10.1016/j.bpj.2018.04.017

3) Mazurov AV, Vinogradov DV, Kabaeva NV, Antonova GN, Romanov YA, VIasik TN, Antonov AS, Smirnov VN (1991) A Monoclonal Antibody, VM64, Reacts with a 130 kDa Glycoprotein Common to Platelets and Endothelial Cells: Heterogeneity in Antibody Binding to Human Aortic Endothelial Cells. Thromb Haemost 66:494–499. doi:10.1055/S-0038-1646445.