Lsr fortessa sorp instrument

DNA was extracted from whole blood using a Roche MagNAPure 96 or Qiagen® DNeasy Blood & Tissue kit according to the manufacturer’s instructions. KIR3DL1 typing was performed using PCR primers described by Boudreau et al. to detect the major alleles *004 (null), *001, *002 (high), *013 (3DS1) and *005, *007 (low). [25 (link)]. Furthermore, flow cytometry was used to determine the KIR3DL1 expression level by staining cells with anti-CD3-APC-H7 (SK7), anti-CD14-APC-H7 (MϕP9), anti-CD16-BV786 (3G8), anti-CD56-BV711 (NCAM1), anti-CD57-BV605 (NK-1), anti-CD19-APC-H7 (SJ25C1; all from BD Biosciences), anti-NKG2C-Alexa Fluor 488 (134,591), anti-KIR2DL1 Alexa Fluor 700 (143211, both R&D Systems), anti-NKG2A-PE (Z199), anti-KIR2DL1/S1-Pe-Cy7 (EB6B), anti-KIR2DL2/L3/S2-Pe-Cy5.5 (GL 183; all from Beckman Coulter), anti-KIR3DL1-APC (DX9; BioLegend). Stained samples were analyzed on a five laser BD LSR Fortessa SORP instrument. Data was analyzed using FACSDiva (v.8.0.1 or later) and FlowJo (v.10.8.1 or later) software (BD Biosciences). The median frequency of KIR3DL1⁺ cells among CD16⁺CD56⁺ NK cells was 11% (range 7.71–56.5%). The HLA-B and -C allele genotype was determined using LABType SSO Class I Locus Typing Tests from One Lambda as described elsewhere [26 (link)]. Complementary KIR ligand typing for HLA-A Bw4 was performed using the Olerup SSP KIR HLA Ligand kit.

Healthy donor PBMCs were thawed and stimulated overnight in 500 U/ml IL-2 (Proleukin, Novartis Pharmaceuticals, Surrey, UK). AML samples were thawed, stained for CD34⁺, and the CD34⁺ blasts were sorted on a 3-laser FACSARIA III flow cytometer (405, 488 and 640 nm; BD Biosciences, San Diego, CA, USA). K562 cells or sorted CD34⁺ AML cells were added to the cultured PBMCs at a ratio 4:1 (PBMC:target cells) and incubated for 4 h in presence of CD107a antibody. After incubation, cells were washed and stained with antibodies directed against CD3, CD56, CD107a, NKG2A, KIR2DL1/S1, KIR2DL2/L3 and KIR3DL1 followed by analysis using a BD LSR Fortessa SORP instrument.

50 μL (2.5 × 10⁶ sperm) of each sperm treatment group was collected at times 0, 1, 2, and 3 h. Samples were washed three times at 300 × g for 5 min with PBS. Subsequently, Zombie UV^TM fixable viability kit (#423107, Biolegend, CA, USA) was used according to the manufacturer's protocol. The samples were fixed for 20 min with 50 μL of 4% (v/v) formaldehyde with 2% BSA. Spermatozoa were centrifuged at 300 × g for 5 min and resuspended in the original volume of PBS. Sperm samples were analyzed using BD LSRFortessa TM SORP instrument (Becton Dickinson, CA, USA) with a 405 nm laser line excitation (50 mW) and a 450/50 nm emission filter. Zombie UV^TM negative spermatozoa were considered viable.

Flow cytometry analyses of the sperm samples were performed using a BD LSRFortessa TM SORP instrument (Becton Dickinson, San Jose, CA, USA). The following lasers and filter parameters were selected, according to which a fluorescent probe was used: FITC blue laser = 488 nm (100 mW) excitation and 530/30 emission filter; propidium iodide and mitotracker red CMXros = 561 nm (50 mW) laser line excitation and 586/15 nm emission filter; and Zombie viability dye and DAPI = 405 nm (50 mW) laser line excitation and 450/50 nm emission filter.
The voltages were set for the optimum resolution, and minimally, 20,000 gated events (based on FSC and SSC) were recorded for each sample. As a standard procedure before each specific sperm parameter evaluation, positive and negative control samples were prepared to ensure the correct setting of the flow cytometer.