Then add 400 ul of 96% ice-cold ethanol and vortex shortly. visual comparison of flow cytometry data in overlay diagrams for myeloid blood cells on various stages of differentiation.? 70% ethanol permeabilization of neutrophils and HL-60 cells results in lower background fluorescence and better peak resolution than MeOH and Saponin permeabilization.? Non-specific antibody Isoliquiritin binding in neutrophils can be efficiently blocked by using 1% BSA and non-immune goat serum. Specifications Table Subject area? Biochemistry, Genetics and Molecular Biology? Immunology and MicrobiologyMore specific subject areaProtein DetectionMethod nameFlow cytometryName and reference of original methodP. O. Krutzik and G. P. Nolan. 2003. Intracellular phospho-protein staining techniques for flow cytometry: Monitoring single cell signaling events. Cytometry, vol. 55?A, no. 2, pp. 61C70.Resource availabilityAnti-GFI1 rabbit antibodies, goat anti-rabbit Mouse monoclonal to CD34.D34 reacts with CD34 molecule, a 105-120 kDa heavily O-glycosylated transmembrane glycoprotein expressed on hematopoietic progenitor cells, vascular endothelium and some tissue fibroblasts. The intracellular chain of the CD34 antigen is a target for phosphorylation by activated protein kinase C suggesting that CD34 may play a role in signal transduction. CD34 may play a role in adhesion of specific antigens to endothelium. Clone 43A1 belongs to the class II epitope. * CD34 mAb is useful for detection and saparation of hematopoietic stem cells Alexa Fluor 488 antibodies (Thermo Fisher Scientific, Waltham, MA, USA) Open in a separate window Method details Reagents 1 RPMI-1640 medium without sodium bicarbonate (Merck, Darmstadt, Isoliquiritin Germany) 2 Sodium bicarbonate (Merck, Darmstadt, Germany) 3 HEPES (Merck, Darmstadt, Germany) 4 PBS tablets without calcium and magnesium (Thermo Fisher Scientific, Waltham, MA, USA) 5 Formaldehyde solution, methanol free (Thermo Fisher Scientific, Waltham, MA, USA) 6 Bovine Serum Albumin, BSA (Merck, Darmstadt, Germany) 7 Fetal Bovine Serum, FBS (Merck, Darmstadt, Germany) 8 Non-immune goat serum (Thermo Fisher Scientific, Waltham, MA, USA) 9 Anti-GFI1 (PA5-77985) rabbit antibodies (Thermo Fisher Scientific, Waltham, MA, USA) 10 Goat anti-rabbit Alexa Fluor 488 antibodies (Thermo Fisher Scientific, Waltham, MA, USA) 11 HL60 cells were purchased from collection of ATCC (Manassas, VA, USA) 12 All-trans-retinoic acid (ATRA) (Merck, Darmstadt, Germany) Additional reagents, used to verify method: 13 Protease inhibitor cocktail cOmplete (Roche Diagnostics, Indianapolis, IN, USA) 14 Phosphatase Inhibitor Cocktail III (Abcam, Milton, United Kingdom) 15 Diisopropylfluorophosphate (DFP) (Merck, Darmstadt, Germany) 16 Z-VAD-FMK (Selleckchem, Houston, TX, USA) 17 Anti-CD66b PE-conjugated antibodies (Becton Dickinson, Franklin Lakes, NJ, USA) Equipment Flow Cytometer, Cytoflex (Beckman Coulter, Brea, CA) Note: This list does not include any small generic laboratory equipment that is assumed to be available. Chemicals and other components could be used from any reliable company. Procedure Human neutrophils isolation and HL-60 Isoliquiritin cell growing 1 Neutrophils were isolated from blood of healthy donors using standard technique with 3% dextran and Ficoll-Paque, which was described previously  and confirmed with flow cytometry analysis of CD66b surface marker, specific for mature neutrophils. (Fig. S1, Supplementary) 2 HL-60 cells were grown in RPMI-1640 medium (with HEPES and sodium bicarbonate) with 10% FBS and 2?mM l-glutamine until concentration 1*106 per ml. To model differentiation process HL-60 cells were treated by 2?mM ATRA according to common used protocols . Differentiation of HL60 was confirmed by CD66b flow cytometry analysis. (Fig. S1, Supplementary) 3 Each experimental sample contained 2*106 cells. Note: Neutrophils could be lost during the sample preparation, so it is better to take a 2C3 times bigger sample. Fixation and permeabilization 4 Resuspend 2*106 cells in 5?ml of PBS containing 0.05% BSA, centrifuge (270? em g /em , 4?C, 6?min). 5 Resuspend the pellet in 1?ml PBS with 2C4% formaldehyde (PFA). 6 Incubate at 37?C for 10?min, then add 5?ml of cold PBS with 0.05% BSA and centrifuge (270? em g /em , 4?C, 6?min). 7 Resuspend the pellet in 1?ml of 70% ice-cold Isoliquiritin ethanol, place on ice for 30?min. Centrifuge (300? em g /em , 4?C, 6?min). Note: Add first 200 ul of cold PBS and resuspend the pellet gently. Then add 400 ul of 96% ice-cold ethanol and vortex shortly. HL-60 cells can be stored at ?20?C for up to 3 weeks after 30?min on ice. Neutrophils cannot be stored this way. We recommend Isoliquiritin proceeding to the next stages of the protocol immediately after the permeabilization of neutrophils. Blocking of non-specific binding of antibodies (ab) 8 Resuspend the pellet in 3?ml PBS with 1% BSA and 10% non-immune goat serum. 9 Incubate 30?min at room temperature (RT), centrifuge (300? em g /em , 4?C, 6?min). Note: For blocking, use the serum of the animal in which secondary Ab were produced. Staining with primary ab 10 Resuspend the pellet in 100 ul of PBS with 1%.