Ack buffer

Manufactured by BD

Sourced in United States

ACK buffer is a laboratory solution used to maintain the pH and ionic strength of biological samples during various experimental procedures. It is a commonly used buffer in molecular biology and biochemistry applications.

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Lab products found in correlation

Dnase 1, by Merck Group (3 mentions) Facscanto 2, by BD (2 mentions) Hepes, by Thermo Fisher Scientific (2 mentions) Complete 1 640 medium, by Solarbio (2 mentions) Collagenase 4, by Solarbio (2 mentions) Streptavidin brilliant violet 605, by BioLegend (1 mentions) Tc10 automatic cell counter, by Bio-Rad (1 mentions) 40 μm cell strainer, by BD (1 mentions) Aria 1 cell sorter, by BD (1 mentions) Dnase 1, by Roche (1 mentions) Liberase, by Roche (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Merck Group (1 mentions) β mercaptoethanol, by Merck Group (1 mentions) Percp cy5.5 anti mouse cd45 antibody, by BD (1 mentions) Ghost dyetm red 780, by Cytek Biosciences (1 mentions) Facsaria 2 cell sorter, by BD (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

ACK lysing buffer ACK lysis buffer

8 protocols using ack buffer

Isolation and Characterization of Avian Immune Cells

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After washing, chunked spleen or longitudinally cut cecal tonsils were minced with the flat end of a 3-ml syringe plunger through a 40-μm cell strainer (BD Biosciences, San Jose, CA) into a 50-ml conical tube (SPL, Pocheon, Korea). To purify immune cells, red blood cells were lysed using ACK buffer (BD Biosciences) for 3 min at room temperature and then washed.
For examination of B cells and macrophages, anti-chicken MHC class II-FITC (clone 2G11), Monocyte/Macrophage-PE (clone KUL01), and Bu-1-Alexa Flour^® 647 (clone AV20) antibodies (all from Southern Biotec, Birmingham, AL) were used. To examine CD4⁺ subtypes of T cells, anti-chicken CD3-Percific Blue (clone CT-4), CD4-FITC, CD8α-SPRD (clone CT-8), TCRgd-PE (clone TCR1), CD5-biotin (clone 2-191) (all from Southern Biotec), and CD25-Alexa Fluor^® 647 (AbD Serotec) antibodies and Brilliant Violet 605 streptavidin (BioLegend, San Diego, CA) were used.
Data acquired by flow cytometry (FACS Canto II, BD Biosciences) were analyzed with FlowJo software (Tree Star, San Carlos, CA). Total cell number was determined by an automatic cell counter TC10 (Bio-Rad, Hercules, CA). The number and proportion of immune cells were calculated.

Lee I.K., Gu M.J., Ko K.H., Bae S., Kim G., Jin G.D., Kim E.B., Kong Y.Y., Park T.S., Park B.C., Jung H.J., Han S.H, & Yun C.H. (2018). Regulation of CD4+CD8−CD25+ and CD4+CD8+CD25+ T cells by gut microbiota in chicken. Scientific Reports, 8, 8627.

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Isolation and Sorting of CD3+CD8+ Cells

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Splenocytes were isolated, and red blood cells were lysed with ACK buffer (BD Bioscience, USA). Cells were then stained with anti-CD3 and anti-CD8 antibodies (Biolegend, USA). CD3+CD8+ cells were then sorted on AriaI Cell Sorter (BD, Bioscience, USA).

Gao H.F., Cheng C.S., Tang J., Li Y., Chen H., Meng Z.Q., Chen Z, & Chen L.Y. (2020). CXCL9 chemokine promotes the progression of human pancreatic adenocarcinoma through STAT3-dependent cytotoxic T lymphocyte suppression. Aging (Albany NY), 12(1), 502-517.

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Lung and Bone Marrow Single-Cell Isolation

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Lung single cells were prepared as described previously with modifications (50 (link)). Briefly, lungs were perfused, minced, and incubated in 100 mg/ml each of Liberase (Roche) and DNAse I (Roche) at 37°C for 1 h in RPMI 1640 medium with 2% newborn calf serum. RBCs were then lysed with ACK buffer (BD Biosciences). A single-cell suspension was prepared by passing the tissue through a 70-mm filter. BM cells were isolated from both hind limbs, and lineage depletion was performed in some experiments using a hematopoietic progenitor (stem) cell enrichment kit (BD Biosciences) according to the manufacturer’s directions, with addition of an anti-CD5 mAb to the lineage mixture.

Kadel S., Ainsua-Enrich E., Hatipoglu I., Turner S., Singh S., Khan S, & Kovats S. (2018). A Major Population of Functional KLRG1– ILC2s in Female Lungs Contributes to a Sex Bias in ILC2 Numbers. ImmunoHorizons, 2(2), 74-86.

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Isolation and Differentiation of Murine Dendritic Cells

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Bone marrow (BM) was isolated from the tibias and femurs of CD11c-YFP mice [46 (link)], as previously described [47 (link)]. Red blood cells were lysed with ACK buffer (BD Biosciences, 555899). BM-derived DCs were cultured in DC medium containing RPMI 1640 (Sigma-Aldrich), 10% FBS (Invitrogen), 1% antibiotic-antimycotic, 15 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine (all from Gibco), 50 µM β-mercaptoethanol (Sigma-Aldrich), and 80 ng/mL GM-CSF (derived from the supernatant of myeloma cells (X63 Ag8.653) transfected with murine GM-CSF cDNA [48 (link)]). Between days 7 and 9, the floating cell fraction containing immature DCs was transferred into tissue-culture-treated dishes (TPP) in DC medium supplemented with 0.1 µg/mL LPS (ALX-581-009-L002, Enzo Life Sciences, Farmingdale, NW, USA) to induce DC maturation. After 24 h, the floating cells were harvested and used for the experiments. Animals were housed and experiments were performed under specific pathogen-free conditions. All experiments were approved by the Cantonal Veterinary Office Zurich under Project License ZH239/19.

Bauer A., Klassa S., Herbst A., Maccioni C., Abhamon W., Segueni N., Kaluzhny Y., Hunter M.C, & Halin C. (2023). Optimization and Characterization of Novel ALCAM-Targeting Antibody Fragments for Transepithelial Delivery. Pharmaceutics, 15(7), 1841.

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Isolation of Lung CD45+ Cells

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Single cell suspensions were isolated from mouse lungs by collagenase digestion following recently published protocols (Reyfman et al., 2019 (link)). Briefly, mice were anesthetized with an overdose of 0.5% pentobarbital, then the lungs were perfused with 1 mL complete 1,640 medium (Solarbio) with 10% FCS (Hyclone) containing collagenase IV (Solarbio) and Dnase I (Sigma) through the trachea, chopped with scissors, and subsequently incubated for 20 min at 37°C with mild agitation. The resulting lung homogenate was passed through a 40-μm filter and resuspended in ACK buffer (BD) for 15 min on ice. Then, the cells were centrifuged at 400 g for 6 min and incubated with Percp-Cy5.5 anti-mouse CD45 antibody (BD) and Ghost Dye^TM Red 780 (Tonbo) for 15 min on ice. BD FACS ARIA II cell sorter was applied to sort CD45⁺ cells from lung cell suspensions pooled from three mice.

Yang H.Q., Wang Y.S., Zhai K, & Tong Z.H. (2021). Single-Cell TCR Sequencing Reveals the Dynamics of T Cell Repertoire Profiling During Pneumocystis Infection. Frontiers in Microbiology, 12, 637500.

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Tissue Dissociation and Single-Cell Preparation

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After euthanasia via CO₂ inhalation, the LNs or spleens were removed, dissociated using a mortar and pestle, digested with 1 μg ml ⁻¹ Liberase TL (Roche, Basel, Switzerland) and 1 mg ml ⁻¹ DNase I (Sigma-Aldrich) in serum free media for 30 min at 37°C. Single-cell suspensions were filtered, counted, and washed in DC media (RPMI 1640 media (Invitrogen) supplemented with 10% heat-inactivated (HI) FCS (Pan Biotech, Germany), 25 mM HEPES, 50 U ml ⁻¹ penicillin (Gibco, MA, USA), 50 μg ml ⁻¹ streptomycin (Gibco), 50 μM 2-mercaptoethanol, sodium pyruvate 1 mM (Sigma), non-essential amino acids at the recommended dilution (Gibco) and 2 mM EDTA (Sigma), before staining for cytometry. When necessary, red blood cell lysis was performed using ACK buffer (BD) for 2 min at room temperature.
Lung tissue was minced and then digested using 5 ml of serum-free RPMI containing 350 enzyme activity units of Collagenase IV (Worthington Biochem, NJ, USA) and 5 mg of DNase I, and incubated for 30 min at 37°C before being passed through a 20 μm filter, washed, counted, subjected to RBC lysis, and then plated similarly to other tissues.

Pejoski D., Ballester M., Auderset F., Vono M., Christensen D., Andersen P., Lambert P.H, & Siegrist C.A. (2019). Site-Specific DC Surface Signatures Influence CD4+ T Cell Co-stimulation and Lung-Homing. Frontiers in Immunology, 10, 1650.

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Isolation and Characterization of Mouse Lung Cells

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Preparation of cell suspensions followed a recently published protocol [16 (link)]. In brief, an overdose of 0.5% pentobarbital was used to anesthetize mice through intraperitoneal injection. Next, collagenase IV (Solarbio) and Dnase I (Sigma) contained in 1 mL complete 1640 medium (Solarbio) with 10% fetal bovine serum (FBS; Hyclone) were infused into the lung through the trachea. Subsequently, the collected lung tissue was chopped with scissors and incubated for 20 min at 37 °C with mild agitation in the 1640 medium with 10% FBS. After filtration through a 40-µm cell strainer (biolegend), the lung homogenate was spun down at 400 g for 6 min and resuspended in ACK buffer (BD) for 15 min on ice to lyse red blood cells.
After washing with PBS, mouse lung cells were stained with fluorochrome-conjugated antibodies using the recommended concentrations according to instructions provided by the manufacturer. Then, the cells were loaded onto FACS Canto II (BD Biosciences, San Jose, CA, USA) and the resulting FACS data were analyzed by FlowJo (version 10.4, TreeStar, Ashland, OR, USA). Table S1 provides the antibodies used in this research.

Yang H.Q., Sun H., Li K., Shao M.M., Zhai K, & Tong Z.H. (2024). Dynamics of host immune responses and a potential function of Trem2hi interstitial macrophages in Pneumocystis pneumonia. Respiratory Research, 25, 72.

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Temporal Immune Response to Poly(I:C)

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The mice were bled at four time points: 1 hr, 4 hr, 8 hr, and 12 hr following the poly(I:C) treatment. The peripheral blood leukocytes were prepared from the blood, and the mice were euthanised to harvest the spleen and the liver. A single-cell suspension was formed of both organs. ACK buffer (BD) was used to lyse the cells, followed by use of Ficoll Histopaque (Sigma-Aldrich, USA) for leukocyte purification. Ficoll was also used to prepare the cells for processing through flow cytometry. Next, the cell monolayer was centrifuged and then separated, and the full count of mononuclear cells per compartment was conducted using a haemocytometer. Finally, cell viability was examined using the trypan blue exclusion assay.

Salem M.L., El-Naggar S.A., Mobasher M.A, & Elgharabawy R.M. (2020). The Toll-Like Receptor 3 Agonist Polyriboinosinic Polyribocytidylic Acid Increases the Numbers of NK Cells with Distinct Phenotype in the Liver of B6 Mice. Journal of Immunology Research, 2020, 2489407.

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