Erratum: Flow Cytometry Analysis of Tissue Factor Expression in Human Platelets

Özet

An erratum was issued for: Flow Cytometry Analysis of Tissue Factor Expression in Human Platelets. The Protocol section was updated.

Özet

This corrects the article 10.3791/67356-v

Protokol

An erratum was issued for: Flow Cytometry Analysis of Tissue Factor Expression in Human Platelets. The Protocol section was from:

4. Sample preparation for flow cytometry analysis of cell surface-associated TF expression in resting and activated platelets

NOTE: For the detection of cell surface-associated TF, use unlabelled αCD142/HTF1 mAb (1 mg/mL) and Alexa Fluor 633 goat anti-mouse IgG as the secondary antibody for signal detection. Adenosine 5'-diphosphate (ADP) is used as a platelet agonist to induce the exposure of TF on the cell surface. A PE-labelled antibody is used as a platelet population marker to minimize spillover, which will be checked by signal compensation.

1. TF staining
   1. Dilute αCD142/HTF1 mAb 1:100 in 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, to obtain a working concentration of 10 µg/mL. Dilute Alexa Fluor 633 goat anti-mouse IgG 1:10 in 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4.
   2. Prepare the three sample tubes and dispense 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, as indicated in Table 3.
   3. Dispense 7.5 µL of diluted αCD142/HTF1 mAb and add 5 µL of ADP (200 µM). Gently invert the citrate vacutainer 5 times to mix the blood and anticoagulant thoroughly, then dispense 5 µL of whole blood into each tube. Gently mix the samples.
   4. Incubate the samples for 20 min at room temperature.
2. Sample fixation
   1. Fix each sample by adding 300 µL of 1% paraformaldehyde (PFA) and incubate the samples for 1 h at room temperature.
      NOTE: Labelled samples fixed with 1% PFA and stored at 4 °C remain stable for up to 4 days.
   2. Add 300 µL of 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, to each tube and centrifuge at 1500 x g for 5 min with brake at room temperature.
   3. Remove the supernatant and resuspend the pellet in 90 µL of 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, by gently pipetting.
3. Staining with the secondary antibody
   1. Dispense 5 µL of diluted Alexa Fluor 633 IgG and 5 µL of αCD41-PE into each tube (see Table 4). Incubate the samples for 15 min in the dark at room temperature.
   2. Add 300 µL of 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, to each tube and mix.
   3. Store the stained samples in the dark until flow cytometry analysis (see step 6).
      NOTE: To calculate the compensation matrix, prepare the samples according to Steps 4.1-4.3 and as outlined in Table 5 and Table 6.

to:

4. Sample preparation for flow cytometry analysis of cell surface-associated TF expression in resting and activated platelets

NOTE: For the detection of cell surface-associated TF, use unlabelled αCD142/HTF1 mAb (1 mg/mL) and Alexa Fluor 633 goat anti-mouse IgG (2 mg/ml) as the secondary antibody for signal detection. Adenosine 5'-diphosphate (ADP) is used as a platelet agonist to induce the exposure of TF on the cell surface. A PE-labelled antibody is used as a platelet population marker to minimize spillover, which will be checked by signal compensation.

1. TF staining
   1. Dilute αCD142/HTF1 mAb 1:100 in 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, to obtain a working concentration of 10 µg/mL. Dilute Alexa Fluor 633 goat anti-mouse IgG 1:10 in 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH  7.4, to obtain a working concentration of 200 μg/mL.
   2. Prepare the three sample tubes and dispense 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, as indicated in Table 3.
   3. Dispense 7.5 µL of diluted αCD142/HTF1 mAb and add 5 µL of ADP (200 μM) to obtain a final concentration of 10 μM ADP. Gently invert the citrate vacutainer 5 times to mix the blood and anticoagulant thoroughly, then dispense 5 µL of whole blood into each tube. Gently mix the samples.
   4. Incubate the samples for 20 min at room temperature.
2. Sample fixation
   1. Fix each sample by adding 300 µL of 1% paraformaldehyde (PFA) and incubate the samples for 1 h at room temperature.
      NOTE: Labelled samples fixed with 1% PFA and stored at 4 °C remain stable for up to 4 days.
   2. Add 300 µL of 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, to each tube and centrifuge at 1500 x g for 5 min with brake at room temperature.
   3. Remove the supernatant and resuspend the pellet in 95 µL of 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, by gently pipetting.
3. Staining with the secondary antibody
   1. Dispense 5 µL of diluted Alexa Fluor 633 IgG and incubate the samples for 15 min in the dark at room temperature.
   2. Add 300 μL of 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, to each tube and centrifuge at 1500 x g for 5 min with brake at room temperature. Remove the supernatant and resuspend the pellet in 95 μL of 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4.
   3. Dispense 5 µL of αCD41-PE into each tube (see Table 4). Incubate the samples for 15 min in the dark at room temperature.
   4. Add 300 µL of 1x PBS without Ca⁺⁺ and Mg⁺⁺, pH 7.4, to each tube and mix.
   5. Store the stained samples in the dark until flow cytometry analysis (see step 6).
      NOTE: To calculate the compensation matrix, prepare the samples according to Steps 4.1-4.3 and as outlined in Table 5 and Table 6.