Storage of samples after permeabilization at?+?4?C and ?20?C in case of neutrophils prospects to significant changes in scatter (for more that 100 instances, storyline C) and decrease of fluorescence level after staining with antibodies (D) according to the Protocol

Storage of samples after permeabilization at?+?4?C and ?20?C in case of neutrophils prospects to significant changes in scatter (for more that 100 instances, storyline C) and decrease of fluorescence level after staining with antibodies (D) according to the Protocol. cell lines (collection HL-60), to study differentiation process and for additional research purposes. We suggest improved technique to analyze and compare nuclear proteins levels in the myeloid differentiation model system (HL-60 cell collection) and / or primary human being neutrophils. This method was justified with measurement of GFI1 protein manifestation level, as well-known transcription element, standard and essential for mature neutrophils. The key protocol features are as follows: ? Suggested protocol allows simply, direct and right visual assessment of circulation cytometry data in overlay diagrams for myeloid blood cells on numerous phases of differentiation.? 70% ethanol permeabilization of neutrophils and HL-60 cells results in lower background fluorescence and better maximum resolution than MeOH and Saponin permeabilization.? Non-specific antibody binding in neutrophils can be efficiently blocked by using 1% BSA and non-immune goat Malathion serum. Specifications Table Subject area? Biochemistry, Genetics and Molecular Biology? Immunology and MicrobiologyMore specific subject areaProtein DetectionMethod nameFlow cytometryName and research of unique methodP. O. Krutzik Malathion and G. P. Nolan. 2003. Intracellular phospho-protein staining techniques for circulation cytometry: Monitoring solitary cell signaling events. Cytometry, vol. 55?A, no. 2, pp. 61C70.Resource availabilityAnti-GFI1 rabbit antibodies, goat anti-rabbit 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, 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 remedy, 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) Products Circulation Cytometer, Cytoflex Malathion (Beckman Coulter, Brea, CA) Notice: This list does not include any small generic laboratory products that is assumed to be available. Chemicals and additional components could be used from any reliable company. Process Human being neutrophils isolation and HL-60 cell growing 1 Neutrophils were isolated from blood POLD4 of healthy donors using standard technique with 3% dextran and Ficoll-Paque, which was explained previously [1] and confirmed with circulation cytometry analysis of CD66b surface marker, specific for mature neutrophils. (Fig. S1, Supplementary) 2 HL-60 cells were cultivated 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 [2]. Differentiation of HL60 was confirmed by CD66b circulation cytometry analysis. (Fig. S1, Supplementary) 3 Each experimental sample contained 2*106 cells. Notice: Neutrophils could be lost during the sample preparation, so it is better to take a 2C3 instances 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 chilly 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 ethanol, place on snow for 30?min. Centrifuge (300? em g /em , 4?C, 6?min). Notice: Add 1st 200 ul of chilly PBS and resuspend the pellet softly. Then add 400 ul.