Issue link: https://ahint.epubxp.com/i/574474
20 ASHI Quarterly Third Quarter 2015 S C I E N T I F I C C O M M U N I C A T I O N S Cell Treatment with Pronase and DNase Donor PBMC or lymphocytes were treated with pronase (4 7 kuntz units/ml; Sigma-Aldrich, St Louis, MO www sigmaaldrich com ) for 15 minutes and DNase (11,000 u/ml; Sigma-Aldrich) for two minutes at 37 o C After treatment, cells were washed twice in 1 ml PBS at 400 x g for one minute Cells were resuspended in 1 ml of FWB, counted and adjusted to a concentration of either 8 3 x10 6 /ml (standard FCXM) or 1 0x10 7 /ml (Halifax FCXM and Halifaster FCXM) Standard FCXM, Tube Method The standard FCXM tube method (Table 1) was performed essentially as previously described (ASHI manual) Briefly, 30 µl of test or control sera and 30 µl (2 5x10 5 ) of enriched donor PBMC were added to 5 ml polystyrene Falcon tubes, mixed by vortexing, and incubated at 4 o C for 30 minutes Cells were washed with 1 ml FWB three times at 500 x g for five minutes Next, 100 µl of antibody cocktail (5 µl of anti-CD3-PerCP, 5 µl of anti-CD19-PE, and 0 25 µl of anti-IgG-FITC in PBS) were added to the cells, mixed by vortexing, and incubated at 4 o C in the dark for 30 minutes Cells were washed twice in 1 ml FWB at 500 x g for five minutes and resuspended in 0 5 ml of FWB before acquisition and analysis FCXM Tray Methods, Standard, Halifax, and Halifaster Protocol Standard FCXM tray method (Table 1) was performed in 96-well, round bottom BD Falcon Microplate trays (BD Biosciences) Briefly, 30 µl of test or control sera and 30 µl (2 5x10 5 ) of enriched donor PBMC were added to each reaction well of the 96-well tray, mixed by vortexing, and incubated at 4 o C for 30 minutes Cells were washed with 200 µl FWB three times at 500 x g for one minute Next, 100 µl of antibody cocktail (5 µl of anti-CD3-PerCP, 5 µl of anti-CD19-PE, and 0 25 µl of anti-IgG-FITC in PBS) were added to the cells, mixed by vortexing, and incubated at 4 o C in the dark for 30 minutes Cells were washed twice in 200 µl FWB at 500 x g for one minute, transferred into 5 ml BD Falcon tubes, and resuspended in 0 5 ml of FWB for acquisition and analysis In FCXM optimization experiments, various FCXM parameters, including serum incubation time, IgG-FITC incubation time, incubation temperature, serum volume, and cell number, were modified as detailed in the results section and figure legends The Halifax FCXM and the Halifaster FCXM protocols were designed based on the results from optimization experiments The differences between the Halifax, Halifaster, and Standard FCXM tray protocols are detailed in Table 1 FCXM Acquisition and Analysis All cellular events were acquired using the FACSCanto II flow cytometer (BD Biosciences, Mississauga, ON, Canada; www bdbiosciences com ) and analyzed with the BD FACSDIVA TM software (BD Biosciences) using the 1024 channel log scale In the optimization experiments, all crossmatch data were expressed as median channel fluorescence shift (MCFS) from the negative control In the validation experiments all crossmatch data were expressed as MCFS from the 3 standard deviation (SD) cutoff The 3SD cutoffs were calculated using the negative patient FCXM MCF values Statistical Analysis Pearson's correlation coefficient was performed using Microsoft Excel software Chi-square analysis was performed using GraphPad Instat software (GraphPad Software Inc , La Jolla, CA; www graphpad com ) Table 1. Comparison of the Standard, Halifax, and Halifaster FCXM Protocols Parameter Standard FCXM Tube Method Standard FCXM Tray Method Halifax FCXM Protocol Halifaster FCXM Protocol Assay platform 5 ml, 12x75 mm tubes 96-well tray 96-well tray 96-well tray Serum volume 30 µl 30 µl 50 µl 30 µl Cell isolation, cell preparation Lympholyte, PBMC Lympholyte, PBMC Lympholyte, PBMC EasySep™ Direct, Lymphocytes Cell volume 30 µl 30 µl 25 µl 15 µl Cell number 2.5x10 5 2.5x10 5 2.5x10 5 1.5x10 5 First incubation time 30 min 30 min 20 min 20 min Washes (1st set) 3x5 min at 500 x g 3x1 min at 500 x g 3x1 min at 500 x g 3x1 min at 500 x g Wash buffer volume 1000 µl 200 µl 200 µl 200 µl Antibody cocktail volume PBS/CD3/CD19/IgG-FITC 100 µl 89.75 µl /5 µl/5 µl/0.25 µl 100 µl 89.75 µl /5 µl/5 µl/0.25 µl 100 µl 94.75 µl /3 µl/2 µl/ 0.25 µl 50 µl 46.88µl /2 µl/1 µl/ 0.125 µl Second incubation time 30 min 30 min 10 min 5 min Washes (2nd set) 2x5 min at 500 x g 2x1 min at 500 x g 2x1 min at 500 x g 2x1 min at 500 x g Final suspension volume 500 µl 500 µl 400 µl 400 µl Total assay time 85 min 65 min 35 min 30 min