Ack lysis buffer

Manufactured by Lonza

Sourced in United States, Switzerland

The ACK lysis buffer is a solution used to lyse red blood cells in biological samples. It effectively disrupts the cell membranes of red blood cells, allowing for the isolation of other cell types, such as white blood cells, for further analysis or experimentation.

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Close spelling variants of this product

ACK buffer (64 citations) Lysis buffer (8 citations) ACK red blood cell lysis buffer (7 citations) Ammonium-Chloride-Potassium (ACK) lysis buffer (6 citations) ACK RBC Lysis Buffer (3 citations) TheraPEAKTM ACK lysis buffer (2 citations) Ammonium chloride lysis (ACK) buffer (2 citations)

211 protocols using ack lysis buffer

Lung and Lymph Node Extraction

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Lungs were removed and minced into smaller pieces in a 6-well plate (Greiner, Germany).
The digestion mixture, composed of 1 mg/mL collagenase D and 0.1 mg/mL DNase I (both from Roche, Switzerland) in complete RPMI medium (Gibco, USA), was added to the samples and incubated for 45 min at 37°C on a lateral shaker. The lung samples were filtered through 100 µm mesh with PBS, washed twice, and the red blood cells were eliminated by ACK lysis buffer (Lonza, USA). Spleens and mediastinal lymph nodes were homogenized by filtering through a 76 µm mesh with ice cold PBS (Lonza), washed twice and red blood cells were eliminated by ACK lysis buffer (Lonza).

Özkan M., Eskiocak Y.C., & Wingender G. (2021). The IL-10^GFP(VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation.

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Isolation of Lung and Lymphoid Cells

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Lungs were removed and minced into smaller pieces in a 6-well plate (Greiner, Germany). The digestion mixture, composed of 1 mg/mL collagenase D and 0.1 mg/mL DNase I (both from Roche, Switzerland) in complete RPMI medium (Gibco, USA), was added to the samples and incubated for 45 min at 37°C on a lateral shaker. The lung samples were filtered through 100 μm mesh with PBS, washed twice, and the red blood cells were eliminated by ACK lysis buffer (Lonza, USA). Spleens and mediastinal lymph nodes were homogenized by filtering through a 76 μm mesh with ice-cold PBS (Lonza), washed twice, and red blood cells were eliminated by ACK lysis buffer (Lonza).

Özkan M., Eskiocak Y.C, & Wingender G. (2021). The IL-10GFP (VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation. PLoS ONE, 16(5), e0247895.

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Isolation and Enumeration of Murine Bone Marrow and Bone Cells

Cited 1 time

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Mice were weighed, and peripheral blood was obtained via submandibular vein puncture. Blood was collected in K2EDTA-coated microtainers, and automated blood count analysis was performed using a Scil Vet abc Plus+ counter (HORIBA Medical, Kyota, Japan). The mice were euthanized by cervical dislocation, and single-cell suspensions of bone marrow and bone fraction were prepared from the hind legs of mice as previously described.^{22 (link)} In brief, hind legs were stripped from soft tissue, and bones were crushed and washed in PBS (Gibco) with 0.5% FCS. Cell suspensions were mechanically dispersed through a 40-µm nylon mesh cell strainer (Corning) and pelleted at 600g, followed by lysis of red blood cells using ACK lysis buffer (Lonza). Cells were subsequently washed and filtered using a 40-µm strainer. To determine bone marrow cellularity, single-cell suspensions, derived from 1 femur, were mixed 1:1 with Trypan Blue (Invitrogen), and the viable cell count was determined using a Countess 3 Cell Counter (Invitrogen). Remnants of crushed bones (bone fraction) were incubated with collagenase for 45 minutes at 37°C, washed with PBS, and filtered through a 40µm strainer. The remaining red blood cells in bone fraction were lysed using ACK lysis buffer (Lonza) and washed again with PBS, followed by another filtration through a 40-µm cell strainer.

Ernst M.P., Pronk E., van Dijk C., van Strien P.M., van Tienhoven T.V., Wevers M.J., Sanders M.A., Bindels E.M., Speck N.A, & Raaijmakers M.H. (2023). Hematopoietic Cell Autonomous Disruption of Hematopoiesis in a Germline Loss-of-function Mouse Model of RUNX1-FPD. HemaSphere, 7(2), e824.

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Cardiac Graft Tissue Dissociation

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Cardiac grafts were perfused with PBS, minced and digested with 1 mg/ml collagenase D (Roche, Indianapolis, IN) and 0.01% DNase I in RPMI medium for 45 minutes at 37°C. Digested suspensions were passed through a 70 µm nylon mesh and centrifuged, cell pellet was suspended in RPMI medium. Single cell suspension were prepared using Lympholyte-M (Cedarlane, Ontario, Canada) density gradient centrifugation as per manufacturers guidelines. Single-cell suspensions were washed with PBS, RBC lysed using ACK lysis buffer (Lonza, Walkerville, MD), and used for the flow cytometry analysis (FACSCantoII or LSR Fortessa, BD Biosciences, Mountainview, CA) or sorting (FACS Aria, BD Biosciences). Spleen, and LN were gently dissociated into single-cell suspensions, and RBC lysed using ACK lysis buffer (Lonza, Walkerville, MD) and used for FACS analysis or FACS sorting.

Lal G., Kulkarni N., Nakayama Y., Singh A.K., Sethi A., Burrell B.E., Brinkman C.C., Iwami D., Zhang T., Hehlgans T, & Bromberg J.S. (2016). IL-10 from marginal zone precursor B cells controls the differentiation of Th17, Tfh and Tfr cells in transplantation tolerance. Immunology letters, 170, 52-63.

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Immune Cell Profiling from Murine Tissues

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Bone marrow cells were isolated by flushing and lysed with ACK lysis buffer (Lonza). Splenocytes were prepared by homogenising whole spleens and passing through 70 μm cell strainers before ACK lysis. Cells were stained with antibodies consisting panels to detect neutrophil/monocyte populations (CD45 (Tonbo), CD11b (eBioscience), CD115 (Biolegend), Ly6C and Ly6G (Tonbo)) and T and B-cell populations (CD8 (eBioscience), CD4 (Biolegend), CD19 (Biolegend), TCRβ (Biolegend)) for 25 min on ice in the dark before washing twice and fixing in paraformaldehyde (2% in PBS) for 15 min at RT. Dead cells were excluded by use of a Zombie NIR Live/Dead fixable dye (Biolegend). The following day, flow cytometry was performed using a FACSCanto flow cytometer (BD Biosciences) and analysed with FlowJo software (FlowJo LLC).

Daniels M.J., Adamson A.D., Humphreys N, & Brough D. (2017). CRISPR/Cas9 mediated mutation of mouse IL-1α nuclear localisation sequence abolishes expression. Scientific Reports, 7, 17077.

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Isolation of Myeloid-Derived Suppressor Cells

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Myeloid-derived suppressor cells were isolated from tissue using the Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol after red blood cell lysis (ACK lysis buffer, Lonza). The purity of the isolated MDSC population was confirmed with flow cytometry to be >90%.

Tavazoie M.F., Pollack I., Tanqueco R., Ostendorf B.N., Reis B.S., Gonsalves F.C., Kurth I., Andreu-agullo C., Derbyshire M.L., Posada J., Takeda S., Tafreshian K.N., Rowinsky E., Szarek M., Waltzman R.J., Mcmillan E.A., Zhao C., Mita M., Mita A., Chmielowski B., Postow M.A., Ribas A., Mucida D, & Tavazoie S.F. (2018). LXR/ApoE activation restricts innate immune suppression in cancer. Cell, 172(4), 825-840.e18.

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B Cell Isolation and Characterization

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BM was collected from WT, Igκ^del, κ-mac, κ-mac Cxcr4^fl/flmb1-cre− or κ-mac Cxcr4^fl/flmb1-cre+ mice and cells were resuspended in staining buffer: (3% (v/v) fetal bovine serum (FBS) in 1x phosphate buffered saline. Erythrocytes were lysed with ACK lysis buffer (Lonza cat # 10-548E) and cells were stained with rat anti-CXCR4 (2B11), rat anti-CD43 (S7), rat anti-IgM (R6–60.2), rat anti-IgD (11 (link)-36 (link)), rat anti-CD19 (1D3) and rat anti-B220 (RA3-6B2l), (all from BD Biosciences) as described previously (77 (link), 78 (link)) and viability dye eFluor 506 (eBioscience). Pre-pro-B cells (CD19−B220+IgM−), pro-B cells (CD19+B220+CD43+IgM−), large pre-B cells (B220+CD43−IgM−FSChi), small pre-B cells (B220+CD43− IgM−FSC^low) and immature B cells (B220+CD43−IgM+) were isolated by cell sorting with a FACSAria II (BD Biosciences).

Okoreeh M.K., Kennedy D.E., Emmanuel A.O., Veselits M., Moshin A., Ladd R.H., Erickson S., McLean K.C., Madrigal B., Nemazee D., Maienschein-Cline M., Mandal M, & Clark M.R. (2022). Asymmetrical forward and reverse developmental trajectories determine molecular programs of B cell antigen receptor editing. Science immunology, 7(74), eabm1664.

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Tracheal Lavage and Cell Analysis

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Mice were sacrificed and a catheter (SR-OX2225CA, Terumo) was inserted into the trachea. The lungs were lavaged and collected cells were treated with ACK lysis buffer (Lonza), and then adhered to a slide by centrifugation. Wright-Giemsa staining was performed using HEMA 3 Stat Pack (Protocol, Fisher).

Lu Q., Yokoyama C.C., Williams J.W., Baldridge M.T., Jin X., DesRochers B., Bricker T., Wilen C.B., Bagaitkar J., Loginicheva E., Sergushichev A., Kreamalmeyer D., Keller B.C., Zhao Y., Kambal A., Green D.R., Martinez J., Dinauer M.C., Holtzman M.J., Crouch E.C., Beatty W., Boon A.C., Zhang H., Randolph G.J., Artyomov M.N, & Virgin H.W. (2016). Homeostatic Control of Innate Lung Inflammation by Vici Syndrome Gene Epg5 and Additional Autophagy Genes Promotes Influenza Pathogenesis. Cell host & microbe, 19(1), 102-113.

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Multi-tissue Cell Isolation and Profiling

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Cells from tumor, spleen, lymph nodes, and lung were mechanically dissociated, and the red blood cells were removed with ACK lysis buffer (Lonza, Allendale, NJ). Cells were blocked with Fc antibody and then labeled with various antibody combinations (Lin/CD45/CD31, Lin/CD45/Sca-1/CD44, CD45/CD11b) (BD Biosciences, San Jose, CA). Data were acquired with the BD LSRFortessa (Becton Dickinson) cell analyzer, and analysis was performed using Flowjo software. Cell sorting was performed using a Becton Dickinson cell sorter.

Hong B., Li H., Zhang M., Xu J., Lu Y., Zheng Y., Qian J., Chang J.T., Yang J, & Yi Q. (2014). p38 MAPK inhibits breast cancer metastasis through regulation of stromal expansion. International journal of cancer. Journal international du cancer, 136(1), 34-43.

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Flow Cytometry of Immune Cells

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Single cell suspensions were made from collagenase IV-digested tumors or spleen by mashing cells through a sterile 70 μM nylon mesh cell strainer using the rubber end of a 3 mL syringe into ice-cold PBS. Red blood cells were removed by treatment with ACK Lysis Buffer (Lonza) for four minutes on ice. Peripheral blood was collected on EDTA-coated tubes and treated with RBC buffer (Biolegend, San Diego, CA) to lyse red blood cells. For detection of surface antigens, the cells were stained with primary antibodies for 30 minutes at 4°C in fluorescence-activated cell sorting (FACS) buffer (0.5% bovine serum albumin, 0.05% sodium azide) in PBS. After the cells were washed, secondary antibodies were added in FACS buffer for another 30 minutes at 4°C. After fluorescent labeling, the samples were washed, acquired with the LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ) and analyzed using FlowJo analysis software (TreeStar, Cupertino, CA). The primary antibodies used were: monoclonal antibodies to mouse CD45 (clone 30F11), mouse CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD11b (clone M1/70) and CD11c (clone N418) or an appropriate negative control (isotype immunoglobulins) (eBioscience). All antibodies were used between 5–10 μg/ml.

Rincón E., Cejalvo T., Kanojia D., Alfranca A., Rodríguez-Milla M.Á., Hoyos R.A., Han Y., Zhang L., Alemany R., Lesniak M.S, & García-Castro J. (2017). Mesenchymal stem cell carriers enhance antitumor efficacy of oncolytic adenoviruses in an immunocompetent mouse model. Oncotarget, 8(28), 45415-45431.

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