High-Throughput Automation With the Attune NxT Autosampler: Consistent Results Across All Wells and Across Plates

• Invitrogen CD45 Mouse Anti-Human mAb, Alexa Fluor  488 Conjugate (Cat. No. MHCD4520)
• Invitrogen CD3 Mouse Anti-Human mAb, APC Conjugate (Cat. No. MHCD0305)
• Invitrogen CD8 Mouse Anti-Human mAb, Pacific Blue  Conjugate (Cat. No. MHCD0828)
• Invitrogen CD4 Mouse Anti-Human mAb, R-PE Conjugate (Cat. No. MHCD0404)
• Attune NxT Flow Cytometer, 4-laser standard configuration (Cat. No. A24858)
• Attune NxT Autosampler (Cat. No. 4473928)
• Invitrogen AbC Total Antibody Compensation Bead Kit (Cat. No. A10497)
• Human whole blood with sodium heparin anticoagulant, collected from a normal donor in compliance with institution-approved policy
• Gibco ACK Lysing Buffer (Cat. No. A1049201)
• Invitrogen Countess II Automated Cell Counter (Cat. No. AMQAX1000)
• Countess Cell Counting Chamber Slides (Cat. No. C10228)
• Invitrogen Trypan Blue Stain, 0.4% (Cat. No. T10282)
• Corning™  Falcon™  96-well U-bottom polystyrene microplates (Fisher Scientific Cat. No. 08-772-54)
• Thermo Scientific Pierce 16% Formaldehyde (w/v), Methanol-Free (Cat. No. 28906)
• Falcon™ 50 mL Conical Centrifuge Tubes (Cat. No. 1443222)
• 12 x 75 mm polystyrene flow cytometry tubes
• Gibco PBS, pH 7.4 (Cat. No. 10010023)
• Gibco AlbuMAX  I Lipid-Rich BSA (Cat. No. 11020021)

The following protocol was used for sample preparation, acquisition, and analysis on the Attune NxT Flow Cytometer. Please see the user guides for detailed instructions on setting up an experiment and running samples [1-3]. Table 1 lists the lasers, detector bandpass filters, and fluorophores used.

Table 1. Instrument configuration and antibody conjugates used.

1. 1. Red blood cell lysis
   1. 1.1 Collect 20 mL of whole blood by venipuncture with sodium heparin anticoagulant.
   2. 1.2 To each of four 50 mL conical tubes, add 5 mL of whole blood and 45 mL of 1X ACK Lysing Buffer.
   3. 1.3 Incubate at room temperature for 10 min.
   4. 1.4 Centrifuge samples at 300 x g for 5 min at room temperature.
   5. 1.5 Aspirate the supernatant, leaving approximately 100 µL to avoid disturbing the cell pellet.
   6. 1.6 Prepare 4% formaldehyde and PBS with 1% BSA for stock reagents.
   7. 1.7 Add 5 mL of PBS with 1% BSA to the cells, and gently mix.
   8. 1.8 Centrifuge samples for 5 min at 300 x g.
   9. 1.9 Aspirate the supernatant, and resuspend the cell pellet in 2 mL of PBS with 1% BSA and gently mix.
   10. 1.10 Combine the samples from all four tubes into one 50 mL conical tube.
   11. 1.11 Count cells on the Countess II Automated Cell Counter as described in the instrument user guide [4], or on another counting device.
   12. 1.12 Dilute cells to a concentration of 1 x 10⁷ cells/mL in PBS with 1% BSA.
2. 2. Antibody labeling and fixation (bulk sample)
   1. 2.1 Add 2 mL of cell suspension prepared in step 1.12 to a new 50 mL conical tube.
   2. 2.2 Add 60 µL of each of the four antibody conjugates, and mix gently.
   3. 2.3 Incubate at room temperature or on ice for 20 min, protected from light.
   4. 2.4 Add 20 mL of PBS with 1% BSA to sample, and gently mix.
   5. 2.5 Centrifuge sample for 5 min at 300 x g.
   6. 2.6 Remove and discard supernatant.
   7. 2.7 Resuspend cell pellet in 20 mL of 4% paraformaldehyde.
   8. 2.8 Incubate at room temperature for 20 min, protected from light.
   9. 2.9 Centrifuge sample for 5 min at 300 x g.
   10. 2.10 Remove and discard supernatant.
   11. 2.11 Resuspend sample in 20 mL of PBS with 1% BSA, and gently mix.
3. 3. Preparation of fluorescence minus one (FMO) controls
   1. 3.1 Add 100 µL of lysed whole blood (from step 1.12 above) to each of five 12 x 75 mm flow cytometry tubes. An optional additional sample of unlabeled cells may be prepared.
   2. 3.2 Prepare FMO controls by adding antibody conjugates and cells to tubes as indicated in Table 2.
   3. 3.3 Incubate at room temperature or on ice for 20 min, protected from light.
   4. 3.4 Add 4 mL of PBS with 1% BSA to each tube, and centrifuge for 5 min at 300 x g.
   5. 3.5 Remove supernatant, and resuspend samples in 1 mL of PBS with 1% BSA.
4. 4. Preparation of single-color compensation control
   1. 4.1 Gently vortex the vials of AbC capture beads (Component A) and negative beads (Component B) for 10 seconds before use [5].
   2. 4.2 Label a sample tube for each of the four antibodies, and add 1 drop of capture beads (Component A) to each tube.
   3. 4.3 Add 5 µL of each individual antibody conjugate to capture beads in the designated tubes (one antibody conjugate per tube), and mix well.
   4. 4.4 Incubate for 15 min at room temperature, protected from light.
   5. 4.5 Add 3 mL of PBS to each tube, and centrifuge for 5 min at 300 x g.
   6. 4.6 Carefully remove the supernatant, and resuspend the bead pellet in 0.5 mL of PBS.
   7. 4.7 Add one drop of negative beads (Component B) to each tube immediately, and mix well.
   8. 4.8 Mix each tube well prior to acquisition by flow cytometry. Perform compensation according to the protocol for the Attune NxT Flow Cytometer using the Negative Gate method. Gate on the bead singlet population on the forward scatter (FSC) vs. side scatter (SSC) dot plot. Record each sample.

Table 2. Preparation of FMO controls.

1. 6. Acquisition
   1. 6.1 Run and record the single-color compensation controls using the automated compensation module. Adjust gates as necessary on the positive and negative populations for each single-color compensation sample. The compensation is set automatically once all the controls have been recorded, and may be viewed as either the compensation matrix or spillover matrix. The spillover matrix shows the amount of spillover from each fluorophore into each of the other fluorescent detectors. Data compensation is calculated using the compensation matrix, which is the inverse of the spillover matrix. In this experiment, minimal spillover is observed (Figure 2).

1. 1. 6.4 Add 200 µL of well-mixed labeled sample (from step 2.11) to each well of the first 96-well plate.
   2. 6.5 Place the 96-well plate in the Attune NxT Autosampler and select “Run Plate”.
   3. 6.6 When acquisition of the first plate is complete, repeat steps 6.4 and 6.5 with the second plate.
   4. 6.7 Adjust the gating using the FMO controls to analyze data (Figure 4).

The combination of fluorophores and antibodies in this experiment were chosen specifically to reduce the amount of compensation. The four fluorophores are each detected by a different laser. Compensation values were determined using automatic compensation and found to be minimal (Figure 2). The FMO controls were useful to ensure optimal placement of gates. Acquisition of each 96-well plate was completed in approximately 43 min. Consistent results were observed across wells on a 96-well plate and between 96-well plates, with coefficients of variation observed below 4% for each population of cells when analyzing percent parent and concentration in cells/µL.

Figures 5–8 show the consistency across each 96-well plate and for all 192 replicate samples for percent positive and concentration of the three cell populations. The CD45+ lymphocytes are a subset of all events, including all debris events. The CD45+ CD3+ CD4+ and CD45+ CD3+ CD8+  populations are gated on the CD45+ lymphocyte gate, and thus represent T cell subsets.

Another method for evaluating consistency is the Heat Map analysis function in Attune NxT Software. The Heat Map view shows a virtual plate layout that represents the wells available on the 96-well plate. The Heat Map Setup panel allows selection of the statistic, gate, and parameter for visualizing the data from the experiment, and definition of the display mode and transition values to analyze the data at a glance. The Heat Map view may be used for analyzing tube-based data in addition to plate-based data. Figure 9 illustrates this visual display, using the concentration (in cells/µL) of the CD45+ CD3+ CD4+ cell population. The selected parameter value for each well is displayed in the corresponding graphic well, and the color is selected by the location of the value on the color transition display.

Automation helps maximize reproducibility between experiments and reduces inter-assay variability and inter-operator variability. With low CVs across and among plates, the Attune NxT Flow Cytometer with Autosampler offers a reliable and high-throughput approach for multiparametric cell screening from 96- and 384-well plates, allowing analysis from smaller sample quantities to maximize efficient use of limited numbers of cells.

This combination of higher-throughput with higher-content flow cytometric analysis of as many as 16 parameters for each cell, with acquisition speeds of up to 1 mL/min, provides a powerful screening platform for drug discovery and systems biology experiments. For further information, read our application note about recommendations for accurate concentration measurements on the Attune NxT Flow Cytometer [6].

1. Attune NxT Software user guide, Pub. No. 100024236, Rev. F.
2. Attune NxT Flow Cytometer user guide, Pub. No. 100024235, Rev. C.0.
3. Attune NxT Autosampler user guide, Pub. No. 100032905, Rev. A.0.
4. Countess II Automated Cell Counter user guide, Pub. No. MAN0014293, Rev. B.0.
5. Roederer M (2002) Compensation in flow cytometry. Curr Protoc Cytom 22:1.14.1–1.14.20.
6. Application note: Recommendations for accurate concentration measurements on the Attune NxT Flow Cytometer.