Versalyse solution

Micro-fragmented lipoaspirate (MLA) specimens were stored overnight at room temperature (RT) protected from light. Samples were then digested with 0.1% collagenase type I in DMEM (both from Sigma-Aldrich, Saint Louis, MO, USA) in a shaking bath at 37°C for 45 minutes. After a 1:2 sample dilution with 2% fetal bovine serum (Biosera, Nuaille, France) in DMEM, the samples were filtered through a 100 μm cell strainer (BD Falcon, Corning, NY, USA) and centrifuged at 300 g for 10 minutes at RT. Supernatants were discarded and cell pellet was resuspended in 1 mL of the VersaLyse solution (Beckman Coulter, Miami, FL, USA). After 10 minutes, the samples were filtered through a 40 μm cell strainer (BD Falcon), centrifuged at 300 g for 10 minutes at RT, and the pellet was resuspended in DMEM. Cells were counted on the Sysmex XT1800 counter (Sysmex, Kobe, Japan).

PB from HCMV-seropositive patients was collected and analyzed between day 0 and day +100 post-HSCT at intervals of 7–10 days or at the next visit at the out-patient clinic. Erythrocytes were lysed using VersaLyse solution (Beckman Coulter, Marseille, France) and resulting peripheral blood mononuclear cells (PBMCs) were stained for 30 min at room temperature (RT) with the antibodies summarized in Table 3.Table 3

Summarized flow cytometry panel used for the monitoring of adaptive NK cells.

Antibody	Conjugate	Clone	Company
CD3	PerCP	OKT3	Biolegend
CD14	PerCP	HCD14	Biolegend
CD56	APC-Cy7	5.1H11	Biolegend
CD16	PE-Cy7	3G8	Biolegend
CD57	FITC	HCD57	Biolegend
NKG2C	PE	134591	R&D
7AAD	–	–	Biolegend
CD8	AF700	HIT8a	Biolegend
NKG2D	BV421	1D11	Biolegend
NKp46	BV605	9E2	Biolegend

Absolute numbers of adaptive NK cells per µl of whole blood were determined using fluorescent beads (FlowCount Fluorospheres™, Beckman Coulter) and absolute cell number were calculated as previously described [42 (link)].
Nine samples (mean: 9; range: 4–12) per patient were analyzed. FACS analysis was performed using FlowJo Version 10 (Treestar, Ashland, USA).

To maximize the cell yield from all three sample types and properly prepare cells for flow cytometry, we equally treated LA, MLA and SVF samples with 1% collagenase type I in Dulbecco’s Modified Eagle Medium (D-MEM) (both from Sigma-Aldrich, Saint Louis, MO, USA) in a shaking bath at 37 °C for 45 min. After 1:2 dilution with 10% heat-inactivated fetal bovine serum (Biosera, Nuaille, France) in D-MEM (Sigma-Aldrich), samples were filtrated through a 100 μm cell strainer (BD Falcon, Corning, NY, USA) and centrifuged at 300× g for 10 min at RT. Supernatants were discarded, and the cell pellet was resuspended in 1 mL of the VersaLyse solution (Beckman Coulter, Miami, FL, USA). After 10 min, samples were filtered through a 40 μm-cell strainer (BD Falcon, Corning, NY, USA), centrifuged at 300× g for 10 min at RT and the cell pellet resuspended in D-MEM (Sigma-Aldrich). The cells were counted on the Sysmex XT1800 counter (Sysmex, Kobe, Japan), and sample volumes were adjusted to contain 3 × 10⁶ cell/mL.

Spleens were carefully dissected from each animal, kept in 1 mL RPMI 1640 (Biochrom, Berlin, Germany) at 0°C, immediately homogenized, filtered (250 μm Nylon Sieve, Alter Chem, Athens, Greece), centrifuged at 1700 rpm for 5 minutes, and resuspended in 1 mL RPMI 1640. Cells were counted using trypan blue and a Neubauer hematocytometer (Poly Optik GmbH, Germany). Splenocytes were incubated in the dark for 15 min with the monoclonal antibodies anti-mouse-CD3 (clone 145-2C11) at the fluorochrome fluorescein isothiocyanate (FITC, emission 525 nm; Cell Lab, Beckman Coulter Inc., Miami, FL, USA); with the monoclonal antibody anti-mouse NK1.1 (clone: PK136) at the fluorochrome phycoerythrin (PE, emission 575 nm; Cell Lab, Beckman Coulter Inc., Miami, FL, USA); and with anti-mouse-CD1d (clone: 1B1) (PE, emission 575 nm; Biolegend, San Diego, CA, USA). Cells were lysed with VersaLyse Solution (Beckman Coulter Inc., Miami, FL, USA), washed with PBS (Merck, Darmstadt, Germany), centrifuged, and resuspended at PBS with 0.16% formaldehyde. Cells were analyzed by flow cytometry using an FC-500 instrument (Beckman Coulter Inc., Miami, FL), with gating for lymphocytes based on their characteristic FS/SS scattering. Cells staining negative for CD3 (−) and positive for NΚ1.1 (+) were considered NΚ cells.

100 µl of blood sample are stained with 5 µl of each antibody for 30 minutes in the dark against CD34-PE (clone 8G12; BD Biosciences, Heidelberg, Germany) CD45-FITC (clone 2D1; BD Biosciences, Heidelberg, Germany) and the viability dye 7AAD (Beckman Coulter Inc., France) applying the CytoFlex flow cytometer (Beckman Coulter Inc., France). Using a lyse/no-wash approach for whole-blood samples, 1 mL of Versa Lyse solution (Beckman Coulter Inc., France) is added and incubated for 15 minutes at room temperature in the dark. Determination of CD34⁺ cells was performed according to the guidelines provided by the International Society of Hematotherapy and Graft Engineering (ISHAGE-protocol).^{20 (link)}

Analysis of cluster differentiation markers was achieved by flow cytometry with a Gallios analytical flow cytometer (Beckman Coulter, Irving, TX, USA). Aliquots (100 µL) of peripheral blood were mixed with the following fluorochrome-conjugated antibodies: Anti-human CD3-APC, CD4-APCeF750, CD8-PC7, CD14-PC5.5, CD16-PB, CD45-KrO, CD54-FITC, and CD56-PE (Beckman Coulter, Irving, TX, USA), and lysed with the Beckman Coulter Versalyse solution following the manufacturer’s recommendations. To obtain the cell counts for the different populations 100 µL of Flow-Count Fluorospheres (Beckman Coulter, Irving, TX, USA) were added.

The SVF was obtained by treating the samples with 1% collagenase type I in D-MEM medium (both from Sigma-Aldrich, Saint Louis, MO, USA) in a shaking bath at 37 °C for 45 minutes, accompanied by a 1:2 dilution with 2% fetal bovine serum (Biosera, Nuaille, France) in the D-MEM medium (Sigma-Aldrich) for stopping the reaction. Filtered through a 100 μm-cell strainer (BD Falcon, Corning, NY, USA), the samples were centrifuged (300 g for 10 min at RT) and the cell pellet was resuspended in 1 mL of the VersaLyse solution (Beckman Coulter, Miami, FL,USA) for a 10-minute incubation. After another filtration through a 40-μm cell strainer (BD Falcon, Corning), the samples were centrifuged (300 g for 10 min at RT), and the cell pellet was resuspended in the D-MEM medium (Sigma-Aldrich). The Sysmex XT1800 hematology analyzer (Sysmex, Kobe, Japan) was used to count the cells.