Standard Operating Procedure. Immunology

Standard Operating Procedure Immunology SOP Ref No SOP title SOP-IU010 PCA Category Laboratory Version 1.0 Page 1 of 12

PCA Introduction The enumeration of fresh or frozen-thawed PBMC (peripheral blood mononuclear cells) is required for use of these cells in many cell-based assays. The number of PBMC in a sample may be counted with the Guava Personal Cell Analyzer (PCA) or by traditional hemacytometer methodology. The Guava PCA system provides a cell counting method that reduces the variability of manual hemacytometer counts, especially when multiple operators are involved. The Guava PCA utilizes the Guava ViaCount assay to allow rapid and reliable determination of cell counts and viability. The ViaCount reagent differentially stains viable and non-viable cells based on their permeability to DNA-binding dyes in the ViaCount reagent. The fluorescence of each dye allows quantitative assessment of viable and non-viable nucleated cells present in a suspension. The system counts the stained nucleated cells and uses the forward scatter (FSC) properties to distinguish free nuclei and cellular debris from cells to determine an accurate cell count. The system automatically performs sample analysis and displays results as the viable and total cell counts per ml and per the original sample volume. The Guava PCA may be used to count each sample in replicate. This system offers a consistent method of counting human PBMC compared to hemacytometer counts, potentially making assays more readily transferable across individuals or laboratories. Equipment and Materials Equipment 1. Guava PCA (Cat. # 0100-1430) (Guava Technologies Inc., 25801 Industrial Blvd. Hayward, CA 94545) 2. Dell Latitude laptop computer (Guava 1000-0390) (Guava Technologies Inc., 25801 Industrial Blvd. Hayward, CA 94545) or equivalent 3. Guava CytoSoft Software CD, Version 2.1.5 (Guava 0500-1030) 4. Vortex mixer (Fisher 12-812), or equivalent 5. Pipetman (Rainin P1000, P200 and P20), or equivalent Page 2 of 12

Materials Reagents a. Guava ViaCount (Guava 1# 4000-0040) (100 tests per bottle), store at 2-8 C, manufacturer's expiry b. Guava Check Kit (Guava 1# 4500-0020) (50 tests), store at 2-8 C, manufacturer's expiry c. Guava Check Beads (Guava # 4200-0070), store at 2-8 C, manufacturer's expiry d. Guava Check Diluent (Guava 1# 4200-0050), store at 2-8 C, manufacturer's expiry e. Guava ICF (Guava 4200-0140), 100 ml bottle, store at room temp, manufacturer's expiry f. Peripheral Blood Mononuclear Cells (freshly prepared from whole blood or thawed from frozen aliquots as described in SOP IU008) g. Sodium Hypoclorite (bleach), any vendor, store at room temperature h. Deionized water from "in house" water tap, or equivalent Supplies a. Sterile tips for pipetman (Costar 4864 and Costar 9032), or equivalent b. Microcentrifuge tubes, 1.5 ml, conical bottom (Fisher 02-681-339), or equivalent c. Screw cap lids with O-ring (Fisher 02-681-366) or equivalent Screw cap lids with O- ring (Fisher 02-681-366) or equivalent note: alternatively regular 1.5ml Eppendorf tubes (Eppendorf 0030 120.086) or equivalent can be used, by cutting the cap with scissors before loading the tube on the GUAVA PCA loader Procedure for GUAVA PCA A. Guava PCA startup 1. Turn on Dell Latitude laptop computer Page 3 of 12

2. Once the computer screen shows the Guava Desktop, turn on the Guava PCA instrument at the back right switch, which activates the green light on the front of the Guava PCA. 3. Open the CytoSoft Version 2.1.5 software application located on the desktop. This turns the laser on. Once CytoSoft has been opened, the laser will stay on until the clean and shut down at the end of the day, even if CytoSoft is closed 4. Warm up the laser for approximately 15 minutes before acquiring samples. If the PCA will not be used for an extended period (6 hours or more), turn off the laser by running Clean and Shut Down. 5. Make sure the waste vial contains bleach and is not full. If the vial is full, empty into the appropriate biohazard container for liquid waste and refill with bleach until the bottom of the vial is completely covered. B. Running Guava Check Run Guava Check at the start of each day to ensure the system is performing properly. Guava Check averages the results from three acquisitions of a standardized Guava Check bead sample to determine if the results are within the expected range. 1. Once initialization of the CytoSoft application is complete, click on the Guava Check program listed in the Main Menu. NOTE: Make sure the system has been warmed up for approximately 15 minutes before acquiring bead samples. 2. Prepare a Guava Check bead sample for acquisition. a. Pipette 950 µl of Guava Check Diluent into a microcentrifuge tube and 100 µl of Guava Check Diluent into a second microcentrifuge tube. The first tube will be used to run the Guava Check; the second tube will be used for setting the threshold for background noise. b. Thoroughly mix the Guava Check Bead reagent using a vortex mixer set at high speed, Open the tube and pipette any volume from the lid of the Guava Check Beads back into the vial. c. Pipette 50 µl of the Guava Check Bead reagent into the first microcentrifuge tube which contains 950 µl of diluent. Cap the tube and vortex at high speed to mix. Vortex immediately before acquiring the sample. 3. Guava Check Page 4 of 12

a. In the Sample Information panel enter the bead lot number and expiration date from the Guava Check Bead reagent vial. b. Enter or verify the Expected Particles/ml value that corresponds to the concentration of beads in the diluted bead sample in the space below the Guava Check lot number. This value is usually 50,000 if prepared at the recommended dilution (a 20-fold dilution of 1 x 10 6 particles/ml). However some Guava check beads lots are prepared to a slightly different concentration and the accurate Expected Particles/ml value can be found stated in accompanying paper within the kit. c. Load the 100 µl Diluent tube onto the sample tube holder. Click on the Adjust Settings button. The software will prompt the loading of diluent. Click "OK" to allow the system to automatically set a threshold to exclude background noise. When a message "Begin a New Data Set appears on the bottom of the screen, the setting is adjusted. d. Confirm the system calibration status by running three replicates of Guava Check bead samples. Mix the Guava Check bead sample thoroughly and load onto the Guava PCA. Click Run 1 st Replicate. The system acquires 1000 events and displays the results in the row for Replicate 1. e. Remove the sample, recap with its original cap, mix well on the vortex and reload on the Guava PCA. Click Run 2 nd Replicate. The system acquires 1000 events and displays the results in the row for Replicate 2. f. Remove the sample, recap with its original cap, mix again and reload on the Guava PCA. Click Run 3 rd Replicate. The system acquires 1000 events and' displays the results in the row for Replicate 3. g. The software automatically calculates the average and % CV for Particles/ml (bead count), FSC and Fluorescence intensities for the three replicates are displayed. If any of the Particles/ml results falls outside the +/- 10% acceptance range, the values appear in red. This calibrates the machine for the day and may take several attempts if there are air bubbles in the tubing. h. If the average of the three replicates is red or % CV is greater than 10%, click on main menu, and run Clean and Shut Down (see Section D in this SOP). Then repeat steps IV.B.3.c-f to rerun Guava Check. If values continue to fall outside the acceptance range, refer to Chapter of the Guava PCA User's Guide for Troubleshooting or call Guava Technologies for service. i. Guava Check may be aborted before all three replicates are run if one of the first two replicates is out of range. In this case run Quick Clean, Backflush or Clean and Shut Down before attempting Guava Check again. Page 5 of 12

j. Once Guava Check has successfully been determined, (see step IV.B.3.g-i) above. load a microcentrifuge tube filled with deionized water and click Quick Clean to rinse the fluid system. Return to the Main Menu when Quick Clean is complete. C. Running the Guava ViaCount Assay to count PBMCs 1. Create a paper batch record from Appendix A to record test sample information. Each sample may be randomly assigned a sample code within an assay to facilitate reference to all sample demographics. That sample code should be indicated for each sample in the assay. The batch record also include space to enter Guava count data and to calculate average cell count for a set of replicates and volume needed to achieve a target cell dilution for sample input in the appropriate assay. 2. Prepare samples for Counting. a. From the CytoSoft Main Menu, click Guava ViaCount for the Cell Count and Viability Assay. b. Prepare a uniform cell suspension of PBMC for counting (freshly prepared from whole blood or by thawing previously isolated and frozen PBMC as described in SOP IU008). c. Label and prepare microcentrifuge tubes for each subject. Pipette 190 µl of ViaCount reagent into each microcentrifuge tube. d. Remove 10 ul of each cell suspension and add to the corresponding, labeled microcentrifuge tube containing ViaCount Reagent for a dilution factor of 20. If more than 1.0 x10 7 cells/ml is suspected in the diluted suspension, start at a higher dilution (reference Guava ViaCount Reagent package insert for recommended dilutions). Vortex gently or flick tube with finger to mix. e. Allow the cells to stain for at least 5 minutes at room temperature. 3. Open a data file, adjust settings and enter sample information. a. Enter ViaCount Reagent lot number and expiration into the sample information screen. b. Click New Data Set to create a data file. Select hard drive location to save file and enter a file name. Click Save. c. Click Settings, then click Adjust Settings. d. A message appears prompting operator to load a stained sample. Vortex a stained sample at moderate speed. Load, and click OK. Page 6 of 12

NOTE: refer to the profile reported in the figure below for correct instrument settings, detailed in the following sections: e. Adjust PM1 and/or PM2 values only if necessary, to position live cells in the upper left of the Viability (PM1) vs Nucleated Cells (PM2) dot plot between 10e0 and 10e1 on the Viability (PM1) axis and between 10e2 and 10e3 on the Nucleated Cells (PM2) axis. f. Adjust the horizontal line (PM2 threshold) on the Viability (PM1) vs Nucleated Cells (PM2) dot plot to separate the viable cells in the upper left from the debris in the lower left. Set the line approximately 2-3mm below the live cell population g. Adjust the vertical line on the Viability (PM1) vs Nucleated Cells (PM2) dot plot to separate viable cells in the upper left from non-viable cells in the upper right. The angle of the line can be adjusted to better discriminate between the two populations. h. FSC threshold may also be adjusted in the FSC vs Viability (PM1) dot plot to eliminate debris, but taking care not to exclude dead cells from the analysys, or the PBMC viability will be overestimated. Page 7 of 12

i. When finished to adjust setting, click Next Step button and enter the data acquisition screen. j. Ensure the Enable EasyFit and Enable Apoptosis Gate buttons in the data acquisition screen are NOT clicked. k. In the Sample information control panel check that dilution factor and original volume are accurate, or modify them accordingly. 4. Run Samples in Guava ViaCount a. Counting samples requires running three replicates of each sample. An optional sample identifier may be entered for the first sample after the first replicate is acquired and before Save and Close Current Sample is clicked. Sample identifiers for all subsequent samples may be added either before or after the sample is acquired. b. Gently vortex the first stained sample on moderate speed and load on the Guava PCA. Click Acquire Next Sample. The system acquires the sample and automatically displays the results. Record Total Viable Cells in Original Sample for this first replicate in a hard copy batch record for the first subject. Click the Save and Close Current Sample button. c. Remove sample, recap with original cap, vortex again and reload on Guava PCA for second replicate. Click Acquire Next Sample. The system acquires the sample and automatically displays the results. Record Total Viable Cells in Original Sample for this second replicate in a hard copy batch record for the first subject. Click the Save and Close Current Sample button. d. Remove sample, recap with original cap, vortex again and reload on Guava PCA for third replicate. Click Acquire Next Sample. The system acquires the sample and automatically displays the results. Record Total Viable Cells in Original Sample for the third replicate in a hard copy batch record for the first. Click the Save and Close Current Sample button. e. Repeat steps b-d for each sample. NOTE: If the concentration is greater than 500 cells/ µl or less than 10 cells/ µl, an error message will appear indicating that counting accuracy may be compromised. Click Abort and remove the sample. Either further dilute or concentrate the sample for accurate counting. f. If any of the triplicate counts is different from the others by +/- 50%, count again with a new aliquot, following steps b-d. g. Run Quick Clean with deionized water in between every 10-15 replicates or more often as needed to prevent build up in the Guava PCA fluid system. Page 8 of 12

h. When data acquisition is complete, close CytoSoft. D. Cleaning of the Guava PCA CytoSoft offers two cleaning cycles, Quick Clean and Clean and Shut Down. It also features Abort and BackFlush functions to clear blockages in the fluid system. 1. Quick Clean: Use this feature of CytoSoft after each assay, in between replicates, or as needed. a. Load a microcentrifuge tube of deionized water or Guava ICF. b. Click Quick Clean to quickly clean the fluid system. c. If the Guava ICF was used, load a microcentrifuge tube of deionized water and click Quick Clean to rinse. If deionized water was used for the Quick clean, cleaning is finished. NOTE: The Guava PCA will prompt you to load a tube of cleaning solution first, but deionized water may be used as a quick flush. d. The tube of water may be left on the Guava PCA until the system is used again or may be discarded if sample acquisition continues. 2. Clean and Shut Down: Use this feature at the end of each day to thoroughly clean the fluid system. Clean and Shut Down also turns off the laser to prepare for system shutdown. a. Click Clean and Shut Down from the main menu. The Guava Clean screen appears. b. Add 10 drops of Guava JCF into a microcentrifuge tube and load onto the Guava PCA. Click Start Cleaning. The laser turns off when Start Cleaning is selected. A message appears to prompt loading of the cleaning solution. Ensure the cleaning tube is loaded and click OK. c. When prompted, load a microcentrifuge tube of deionized water. Click OK. d. A message will appear End of Cleaning. Click Main Menu to return to the CytoSoft main menu or click Exit to close CytoSoft. e. If continuing to run samples (i.e., Clean and Shut Down was run after Guava Check failure or in between assays), allow the laser to warm up for at least 5 minutes. NOTE: Never leave Guava ICF or any cleaning agent on the Guava PCA overnight or for an extended period of time. Always leave a tube of deionized water after cleaning. Page 9 of 12

3. Purging the Fluid System: The BackFlush feature reverses the flow of fluid out of the flow cell. a. If a blockage or clog is suspected due to a very low cell count or no cell counts at all, click Abort. b. Click BackFlush. A message instructs to "load a tube of 20% bleach for cleaning". Load a tube of water or Guava ICF and click OK. c. If the Guava ICF was used, load a tube of deionized water and click Quick Clean to rinse any residual cleaning agent. If deionized water was used, continue sample acquisition. d. Replace the sample and click Acquire Next Sample. 4. Shut Down of the instrument and computer. a. Once sample acquisition is complete for the day and Clean and Shut Down has been performed, click Exit in the Main Menu to close the software. b. Shut down the computer and the Guava PCA. E. Analysis A. Refer to batch record created in section C. The counts for each sample and for each replica will be represented in the batch record. B. Alternatively, the GUAVA Excel Utility Program can be used to extract pertinent analysis information from a ViaCount file and create an Excel spreadsheet file. This procedure avoids the need for filling the batch record during acquisition. Counting data can be entered in the batch record at the end of the analysis. 1. Once the ViaCount program has been closed, double click on the GuavaExcelUtil.exe icon on the desktop. 2. Choose the CSV file created during acquisition from the dialog box. If a dialog box appears indicating that the document contains macros, select the Enable Macros option. 3. The utility program creates an Excel workbook with the appropriate information for listed for each sample (replicas) counted. The file name is the same as the CSV file but with the.xls extension. The original CSV file is unchanged. 4. The file can be printed and cell counts can now be entered in the batch record. Page 10 of 12

Calculations are performed as follows: 1. The three replicates of Total Viable Cells in Original Sample acquired for each sample must be averaged with the following formula: Average = (Replicate 1 + Replicate 2 + Replicate 3)/3. 2. Volume to resuspend cells (ml) should be calculated based on the Average of the three replicates. Volume to Resuspend Cells (ml) = Average of three replicates (cells)/desired cell concentration (cells/ml) Page 11 of 12

Appendix A: Batch Record of Samples Counted (make copies and use one new sheet for each assay set-up) Assay information assay date Operator Sample key sample code 1a 1b 1c 1 average R10 ml for 1 2a 2b 2c 2 average R10 ml for 2 3a 3b 3c 3 average R10 ml for 3 4a 4b 4c 4 average R10 ml for 4 subject identification clinical site Sample Timepoint PBMC date Total viable cell count (triplicate) + average R10 ml for targeted cell concentration SIGNATURE DATE Page 12 of 12