Nexcelom Bioscience
Platelets Cellometer Applications

Platelet Counting Using Cellometer Image Cytometry

Compatible with Cellometer:

Fluorescent Detection of Platelets Using Calcein AM

Calcein-AM (Calcein acetoxymethyl ester) is a cell permeable, non-fluorescent compound. Upon crossing the cell membrane, calcein-AM is rapidly hydrolyzed by cellular esterases inside live cells. The hydrolysis cleaves the AM group, converting the non-fluorescent calcein-AM to a strongly green fluorescing calcein. Because the created calcein more hydrophilic it is trapped inside the cells with intact membranes. Cells that do not possess active cytoplasmic esterases are unable to convert calcein-AM to calcein, and therefore do not fluoresce green. This allows for a quick and easy detection of metabolically-active cells in a sample.

Calcein AM

Calcein AM

Hydrophobic (readily absorbed by cells)

Endogenous Intracellular Esterases

Endogenous Intracellular Esterases

Calcein

Calcein

Hydrophilic (remains in the cytosol of cells with intact membranes

Since calcein-AM does not require DNA binding, it stains all metabolically-active cells and can be used to measure metabolic activity in non-nucleated cells, such as platelets1 2. Because calcein-AM is photostable, has low cytotoxicity, and does not affect cellular functions, it is a popular stain for the examination of cell vitality and metabolic activity.

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Assay Protocol for Platelet Measurement in Whole Blood

Preparation of Calcein AM Reagent

  1. Pipette 2 µl Calcein-AM into 18 µl of dH2O. This is now Calcein-AM Solution A. Mix by pipetting up and down at least 15 times.

Staining Procedure for Platelets

  1. Dilute the platelet sample by adding 5 µl of platelet sample into 85 µl of 1x PBS. Mix well by pipetting up and down.
  2. Add 5 µl of Calcein-AM Solution A to 45 µl of the diluted platelet sample.
  3. Gently pipette the sample up and down ten times, then incubate for 20 min at 37°C in the dark.
  4. After the 20 minute incubation, the sample is ready for analysis.
  5. Gently mix the cell sample by pipetting up and down at least ten times, then load 20 µL into the Cellometer counting chamber and insert into the Cellometer instrument.
  6. Wait 60 seconds for the cells to settle in the chamber
  7. Type a name for your sample into the Sample ID text box
  8. Set the dilution factor to 20.

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Detection of Enzymatically Active Platelets Stained with Calcein AM

Bright field image of a diluted whole blood sample

Bright Field image of a diluted whole blood sample

Fluorescent image of Calcein AM stained cells

Fluorescent image of Calcein AM stained cells

Fluorescent counted image of Calcein AM stained cells

Counted fluorescent image
total number of counted cells calcein-AM active concentration of cells

The vitality of the cell sample is measured by staining the cells with calcein-AM. Once the counting is complete, the Cellometer software automatically reports the total number of counted cells that are calcein-AM active, and the concentration of cells. Displayed above are the captured bright-field, captured fluorescent, and the counted fluorescent images from a whole blood sample. Only the white blood cells and platelets are stained with calcein-AM. Since red blood cells are not stained, they do not interfere with counting. In the counted fluorescent image, counted particles are outlined with a green circle. In this case only the platelets are counted whereas the white blood cells, outlined with a yellow circle, are excluded from counting based on their cell size.

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Cellometer Instruments for Platelet Measurement: Vision 10x, Auto 2000, and X2

Cellometer for platelet measurement

With the Cellometer Vision 10X, Auto 2000, and X2 just 20 µl of sample is added to the Cellometer Counting Chamber. Imaging and analysis of the samples is completed in less than 30 seconds. Bright field and fluorescent cell images can be viewed to check cell morphology and verify cell counting. Total cell count, concentration, and mean diameter are automatically displayed.

Pipette 20 µl of cell sample into a disposable counting slide

1. Pipette 20 µl of cell sample into a disposable counting slide.

Insert slide in to cell counter

2. Insert slide into the instrument

select calcein AM platelet assay

3. Select assay from a drop down menu

platelet results cell count, concentration, and diameter

4. Click count, acquire image and view cell count, concentration, diameter

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Platelet Bright Field Detection and Concentration Measurement

Laboratories that isolate and work with purified platelet samples can measure the platelet concentration using bright-field imaging. This assay is only recommended for purified platelet samples without red blood cells or other contaminating cellular debris, including platelet-rich plasma (PRP).

Assay Protocol for Purified Platelet Measurement

Sample Preperation
  1. Dilute the platelet sample by adding 5 µl of platelet sample into 95 µl of 1x PBS. Mix well by pipetting up and down.
  2. Gently mix the cell sample by pipetting up and down at least ten times, then load 20 µL into the couting chamber.
  3. Wait 60 seconds for the cells to settle in the chamber.
  4. Type a name for your sample into the Sample ID text box.
  5. Set the dilution factor to 20.

Measuring Platelet Concentration Using Bright Field Imaging

Bright field of purified platelets

Bright field cell image of purified platelets

Counted bright field image

Counted bright field cell image of purified platelets

Purified platelets assay results

Purified platelets assay results

Shown above are two bright-field images of purified platelets. The image on the left was captured using a Cellometer 10x instrument. Multiple images are captured and automatically analyzed. Counted cells are outlined in green and the total cell count, concentration, and mean diameter are automatically displayed.

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Cellometer Instruments for Platelet Measurements: Vision 10x, X1 and X2

There are multiple Cellometer instruments that are capable of performing platelet bright-field counting. With the Cellometer X1, X2, and Vision 10x just 20 µl of sample is added to the Cellometer Counting Chamber. Imaging and analysis of the samples are completed in less than 30 seconds. Bright field cell images can be viewed to check cell morphology and verify cell counting. Total cell count, concentration, and mean diameter are automatically displayed.

Pipette 20 µl of cell sample into a disposable counting slide

1. Pipette 20 µl of cell sample into a disposable counting slide.

Insert slide in to cell counter

2. Insert slide into the instrument

select purified platelets assay

3. Select assay from a drop down menu

cell count, concentration, and diameter

4. Click count, acquire image and view cell count, concentration, diameter

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Conclusions

Platelet counting using Cellometer image cytometry is ideal for platelet containing clinical samples. The ability of the instruments to measure platelet concentration in less than 30 seconds improves counting efficiency and consistency. Platelets can be stained with Calcein AM to determine sample vitality and measure the concentration of enzymatically active platelets in a single assay. To determine which instrument is best for you, contact a Nexcelom specialist today.

Give us a call Give us a call 978-327-5340. Experienced Nexcelom Applications Specialists are available 8:30am to 5:30pm EST to assist with selection of a Cellometer system.
Ask an Applications Specialist

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Publications Using Cellometer for Platelet Analysis

  • Sa Q and Hoover-Plow JL. (2011) EMILIN2 (Elastin microfibril interface located protein), potential modifier of thrombosis. Thrombosis Journal 9(9): 1-8
  • Panigrahi S, Ma Y, Hong L, et al. (2012) Engagement of Platelet Toll-Like Receptor 9 by Novel Endogenous Ligands Promotes Platelet Hyperreactivity and Thrombosis. Circulation Research 112(1):103-12
  • Zhao Y, Malinin NL, Julia Meller J et al. (2012) Regulation of Cell Adhesion and Migration by Kindlin-3 Cleavage by Calpain. The journal of biochemical chemistry 287(47):40012-20

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Publications Utilizing Calcein AM for Platelets Analysis

  1. Morrell CN, Matsushita K, Chiles K et al. (2005) Regulation of platelet granule exocytosis by S-nitrosylation. PNAS 102(10):3782-7
  2. Verheul HMW, Jorna AS, Hoekman K, et al. (2000) Vascular endothelial growth factor−stimulated endothelial cells promote adhesion and activation of platelets. Blood 96(13):4216-21

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