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Performing a Four-Part Differential, with Cell Concentrations for Human Peripheral Blood Populations, on the Accuri® C6 Flow Cytometer System®
Sponsored,vendor-submitted protocol   Published in November 2009 (p.17) DOI: 10.2144/000112999 Abstract

The Accuri C6 Flow Cytometer System is an ideal platform on which to perform no-wash, differential analysis of human peripheral blood samples. The fixed-voltage PMTs simplify data collection and reduce the potential for data loss due to improper signal amplification. The broad dynamic range allows varied-size peripheral blood populations to be easily analyzed in the same data file. The Zoom Tool feature of the CFlow® Plus software gives the user precise control when setting gates and allows calculation of the cell concentration per µL of original peripheral blood for platelets, lymphocytes, monocytes, granulocytes, and eosinophils (Figure 1).

The Accuri C6 Flow Cytometer System is an ideal platform on which to perform no-wash, differential analysis of human peripheral blood samples. The fixed-voltage PMTs simplify data collection and reduce the potential for data loss due to improper signal amplification. The broad dynamic range allows varied-size peripheral blood populations to be easily analyzed in the same data file. The Zoom Tool feature of the CFlow® Plus software gives the user precise control when setting gates and allows calculation of the cell concentration per µL of original peripheral blood for platelets, lymphocytes, monocytes, granulocytes, and eosinophils (Figure 1).

Materials

Accuri C6 Flow Cytometer System (Accuri Cytometers, Inc.)

Blood Collection Tube with Anticoagulant

Directly Conjugated Antibodies





Sample Preparation

  1. Collect blood in standard anti-coagulant tube. Keep at RT, rocking gently, until ready to stain.

  2. Aliquot 50–100 µL of whole peripheral blood into standard 12 × 75 mm or 1.5 mL tubes.

  3. Add appropriately diluted, directly conjugated antibodies following an experimental plan that includes single-stained controls.

  4. Stain cells for 20 min at RT, in the dark, gently shaking.

  5. Add red cell lysis buffer at a volume 10-fold greater than the initial volume of whole blood.

  6. Vortex each tube, gently, after adding lysis buffer. Place tubes in dark for 10 min.

Data Collection on the C6

  1. Power on the C6, check performance, and perform a Fluidics Calibration cycle.

  2. Open the CFlow Plus Template "HPB 4 Part Differential."

  3. Verify instrument settings. Refer to full-length protocol.

  4. Using an isotype control tube collect 200,000 total events and PAUSE.

  5. Adjust R1 on the FITC vs. PE-Cy7 plot to contain between 0–5% background events.

  6. Using a CD45-PE-Cy7 positive tube collect data into a new well. Allow the C6 to stop automatically when it reaches the run limit of 100,000 events in R1.

  7. Re-adjust R1, if needed, to encompass the CD45-PE-Cy7+ population.

  8. Collect data for all remaining tubes, allowing the C6 to stop automatically on a run limit of 100,000 events in R1.

Data Analysis

  1. Verify fluorescence compensation values.

    1. Open the compensation matrix.

    2. Calculate median values for data in wells A1–A5 and copy into the appropriate cells of the C-Comp Worksheet.

  2. Verify the location of the five populations by backgating on markers specific for each population to the CD45-PE-Cy7 vs. SSC plot. Use the Zoom Tool to precisely adjust the population gates.

    1. Platelets: Gate CD41-FITC+, CD45-PE-Cy7-cells and adjust gate P4.

    2. Lymphocytes: Gate CD3-FITC+, CD45-PE-Cy7+ cells and adjust gate P5.

    3. Monocytes: Gate CD11b-PE+, CD14-FITC+ cells and adjust gate P6.

    4. Granulocytes: Gate CD11b-PE+, CD14-FITC-cells and adjust gate P7.

    5. Eosinophils: Gate high-green auto-fluorescence, high SSC population in gate P1 and adjust gate P3.

    6. Use the Zoom Tool in CFlow to aid in drawing precise non overlapping gates on CD45 vs. SSC plot (Figure 2, b and c).

  3. Calculate each population per sample tube and per 100 µL of original blood sample (Table 1).

    1. Copy and paste the data columns for gate label, cell number and volume from the CFlow Plus statistics table into a spreadsheet program.

    2. Calculate the number of cells in each population per tube, and then per volume of original blood sample.









Correspondence

Address correspondence to Accuri Cytometers, Inc., 173 Parkland Plaza, Ann Arbor MI 48103; Tel.: (734) 994-8000; www.AccuriCytometers.com, [email protected]

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