Anti cd34 apc

Cells of Stage I (PDL 65 or 70), Stage II (PDL 92), Stage III (PDL 200) and Stage IV (PDL 252) were harvested with trypsin and suspended in 5% FBS-PBS at the concentrations of 1–2 x 10⁶ cells/ml. 100 μl of each cell suspension was mixed with 5 μl of antibody diluted to 20 fold with 5% FBS-PBS. After incubation at 4°C for 20 min, cell suspension was washed 2 times with 5% FBS-PBS and was suspended with 500 μl of 4% paraformaldehyde–PBS. Data acquisition of 10,000 cells was performed using FACSCanto (BD). Analysis was performed using FlowJo software (TOMY Digital Biology). Antibodies used in this test were as follows, APC anti-CD34 (555824, BD), FITC anti-CD44 (IM1219U, Immunotech), FITC anti-CD45 (F0861, DAKO), RPE anti-CD73 (550257, BD), FITC anti-CD90 (F7274, DAKO), and RPE anti-CD105 (A07414, Immunotech). APC IgG1 (X0968, DAKO), FITC IgG1 (X0927, DAKO), RPE IgG1 (X0928, DAKO), and RPE IgG3 (731609, Immunotech) were also used as negative control.

Cells from dissociated EB were filtered through 50 μm filter (Partec) before stained for FACS analysis. All antibodies were purchased from BD Bioscience, including anti-CD34-APC (#555824), anti-CD43⁻FITC (#555475), anti-CD31-PE (#555446), anti-CD31-FITC (#555445), anti-CD73-PE (#550257), anti-CD184- PE-Cy7 (#560669), and anti-CD309- Alexa Flour 647 (#560495). Cells were stained for 30 minutes at 4 degree in the dark then washed twice with PBS supplemented with 2% FBS before flow cytometry. DAPI was used for selection of live cells. All antibodies for flow cytometry were diluted at 1:5 ratio.

CD34⁺ cells from ABM and UCB were enriched using the Human CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec) according to manufacturer instructions and our previous reports [9 (link)]. After staining with PE-conjugated antilineage markers (CD3, CD8, TCR, CD56, CD14, CD11b, CD20, CD19, and CD235a), anti-CD34-APC, anti-CD38-FITC, and anti-CD45RA-PE-TxR conjugated antibodies, primitive cell populations were highly purified by multicolor flow cytometry using a FACSAria sorter (BD Biosciences). Hematopoietic stem cells (HSC) were separated as Lin⁻CD34⁺CD38⁻CD45RA⁻, while multipotent progenitors (MPP) were sorted as Lin⁻CD34⁺CD38⁺CD45RA⁻, and early lymphoid progenitors (ELP) as Lin⁻CD34⁺CD38⁺CD45RA⁺, as described [9 (link)]. Upon harvesting from culture, anti-CD56-PE, anti-CD11c-FITC, and anti-CD16-APC (BD Biosciences) were used to evaluate innate cell production in lymphoid lineage conditions. NK cells were identified by flow cytometry in a FACSCanto II equipment (BD Biosciences) as CD56⁺CD11c⁺ or CD56⁺CD16⁺CD11c⁺ cells, while dendritic cells (DC) were detected as CD56⁻CD11c⁺. Analysis of flow cytometry data was performed using the FlowJo 10 software (TreeStar Inc., USA).

The levels of ex vivo Per2 expression was evaluated using whole blood 24 h after draw. Two hundred microliter of whole blood was stained for 20 min at 4°C with surface antibody anti-CD34 APC and anti-CD45 V500 (Becton-Dickinson BD); following monoclonal staining, BD Pharm Lyse (BD Biosciences) lyse solution was used to lyse red blood cells according to standard procedures and fixed with 4% paraformaldehyde. After red cell lysing, cells were incubated with anti-PER2 antibody (ab179813 Abcam) or anti-Sirt1 Alexa Fluor 488 (ab196368 Abcam) for intracellular staining 20 min at room temperature using a permeabilizing buffer containing 0.5% saponin (PBS1X /BSA 1%/NaN3 0.1%/Saponin 0.5%) and then washed with 0.1% saponin buffer. Alexa Fluor 488-coniugated goat anti rabbit (Invitrogen) diluted in staining buffer containing 0.1% saponin was used as secondary antibody. To evaluate cell viability, we used the 7AAD staining. In some experiments 1 mL of whole blood of HIV infected patients was stimulated with different concentrations of Resveratrol (R5010- Sigma-Aldrich) and YK-3-237 (Tocris) overnight at room temperature (see section results for details). After stimulation, the frequency of HPC expressing Per2 was evaluated by flow cytometry. At least 300,000 total cell events were acquired on FACSCanto II (Becton-Dickinson BD) flow cytometer and analyzed using DIVA software.

Cells were isolated from the lungs using a GentleMACS dissociator (Miltenyi Biotec) according to the manufacturer’s protocol. Briefly, lungs were placed in GentleMACS C tubes (Miltenyi Biotec), mixed with DNase I and collagenase D, and homogenized. The fragments were incubated at 37 °C for 30 min under slow mixing and homogenized again. The homogenized lung was passed through a 70-μm pore-size cell strainer, and red blood cells were removed using a red blood cell lysis buffer. Cells were then stained as described previously using the following antibodies: anti-CD11c-PE (eBiosciences) and anti-CD11b-FITC (eBiosciences).
Human PBMC were treated and analyzed as previously described58 (link). The following monoclonal antibodies were used: anti-CD31 FITC (BD Pharmingen), anti-CD34 APC (BD Pharmingen), anti-CD45 PE-Cy5 (eBiosciences), and anti-VEGFR2 PE (R&D Systems). Isotype controls were all purchased from BD Biosciences. Fluorescence was analyzed using FACScalibur and CellQuest Pro software (BD Biosciences).

Apoptosis evaluation was performed by an Annexin V binding assay using LSR Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cells were treated with various concentrations of venetoclax with or without ATO for 48 h and resuspended in Annexin V binding buffer. They were then incubated with Annexin V-FITC (BD Biosciences) and propidium iodide (PI) or 7-AAD (Beckman Coulter, Brea, CA, USA) for 15 min before flow cytometry analysis. To examine the apoptosis in the CD34⁺CD38⁻ cell fraction, cells were stained with anti-CD34-APC (BD Biosciences), anti-CD38-PE (BD Biosciences), and 7-AAD (Beckman Coulter) for 30 min. The labelled cells were subsequently resuspended in Annexin V binding buffer and incubated with Annexin V-FITC (BD Pharmingen) for 15 min before flow cytometry analysis. Data were analysed using the FACSuite software (BD Biosciences).