Ack lysis buffer

Manufactured by Merck Group

Sourced in United States

ACK lysis buffer is a solution used to lyse red blood cells, also known as erythrocytes. It is commonly used in sample preparation for flow cytometry and other cell analysis techniques that require the removal of red blood cells from a sample.

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Lab products found in correlation

40 μm cell strainer, by BD (9 mentions) Automacs, by Miltenyi Biotec (4 mentions) Collagenase d, by Roche (3 mentions) Facsaria, by BD (3 mentions) Dnase 1, by Merck Group (2 mentions) Mouse ifnγ elisa kit opteia, by BD (2 mentions) Percoll, by GE Healthcare (2 mentions) Paramagnetic bead conjugated anti cd11c, by Miltenyi Biotec (2 mentions) Soluble anti mouse cd3, by BD (2 mentions) Milliplex kit, by Merck Group (2 mentions) 70 μm nylon mesh, by BD (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Tc20 automated cell counter, by Bio-Rad (2 mentions) Streptavidin conjugated pe or apc, by Thermo Fisher Scientific (2 mentions) Facs aria 1 or 2, by BD (2 mentions) Countbright absolute counting beads, by Thermo Fisher Scientific (2 mentions) Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Collagenase type 1, by Merck Group (1 mentions) Collagenase type xi, by Merck Group (1 mentions)

Close spelling variants of this product

Ammonium-chloride-potassium (ACK) lysis buffer (4 citations) ACK buffer (13 citations) Lysis buffer (324 citations)

46 protocols using ack lysis buffer

Isolation of Single Cell Suspensions from Thoracic Aorta, Lung, and Lymphoid Tissues

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Single cell suspensions from thoracic aorta were prepared by enzymatic digestion as previously described with minor modifications (36 , 37 (link)). Briefly, thoracic aorta segments were dissected following vasculature perfusion with 2mM EDTA buffer(5ml), PBS (10ml), and FACS buffer (10ml) respectively. Thoracic aorta, segment above diaphragm, was cut into small pieces and digested in 3ml enzyme cocktail containing 400 U/ml collagenase type I (Millipore Sigma, #C0130), 120 U/ml collagenase type XI (Millipore Sigma, #C7657), 60 U/ml hyaluronidase type I (Millipore Sigma, #H3506), and 60 U/ml DNase I (Millipore Sigma, #11284932001) in RPMI supplemented with 10% heat-inactivated fetal bovine serum (hiFBS) for 50 minutes at 37°C. Digested products in suspensions were filtered through a 40 μm cell strainer (BD Falcon, San Jose, CA) followed by red blood cell (RBC) lysis (ACK lysis buffer, Millipore Sigma) for 3 min at room temperature.
Single cell suspensions from mouse lung, draining lymph nodes, or spleen were prepared by mechanically mincing collected organs though a 40 μm strainer followed by RBC lysis for 3 min at room temperature as described (38 (link)).

Gu B.H., Sprouse M.L., Madison M.C., Hong M.J., Yuan X., Tung H.Y., Landers C.T., Song L.Z., Corry D.B., Bettini M.L, & Kheradmand F. (2019). A novel animal model of emphysema induced by anti-elastin autoimmunity. Journal of immunology (Baltimore, Md. : 1950), 203(2), 349-359.

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Comprehensive Immune Profiling Protocol

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Serum was collected by cardiac puncture. BAL samples were collected by tracheal catherization and lavage with 1 ml PBS + protease inhibitor cocktail (Roche, Mannheim, Germany). Spleens were placed into C-tubes and homogenized via gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). After filtration and red blood cell lysis (ACK lysis buffer, Millipore Sigma, St. Louis, MO), 1 × 10⁶ cells were plated in 200 μl RPMI-10% FBS for antigen restimulation then supernatants harvested after 72 h culture for cytokine analyses.

Valli E., Harriett A.J., Nowakowska M.K., Baudier R.L., Provosty W.B., McSween Z., Lawson L.B., Nakanishi Y, & Norton E.B. (2019). LTA1 is a safe, intranasal enterotoxin-based adjuvant that improves vaccine protection against influenza in young, old and B-cell-depleted (μMT) mice. Scientific Reports, 9, 15128.

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Cytokine Secretion Assay for Immune Cells

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Spleens and lymph nodes were immediately excised from euthanized C57Bl/6 or OT-I mice and dissociated in vitro to achieve single-cell suspensions. Red blood cells were lysed with ACK lysis buffer (Sigma-Aldrich) for 2 min. Cells were resuspended at 1 × 10⁶ cells/mL in Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco) supplemented with 5% FBS, 1% penicillin–streptomycin, 40 μmol/L 2-Mercaptoethanol. Cells were cocultured with target cells as described in the text. Cell-free supernatants were then collected 72 h later and tested for IFNγ (Mouse IFNγ ELISA Kit; OptEIA, BD Biosciences) production by ELISA as directed in the manufacturer’s instructions.

Driscoll C.B., Schuelke M.R., Kottke T., Thompson J.M., Wongthida P., Tonne J.M., Huff A.L., Miller A., Shim K.G., Molan A., Wetmore C., Selby P., Samson A., Harrington K., Pandha H., Melcher A., Pulido J.S., Harris R., Evgin L, & Vile R.G. (2020). APOBEC3B-mediated corruption of the tumor cell immunopeptidome induces heteroclitic neoepitopes for cancer immunotherapy. Nature Communications, 11, 790.

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Generating Bone Marrow-Derived Dendritic Cells

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Bone marrow-derived dendritic cells (BMDCs) were generated according to a modified version of a previously described method(Helft et al., 2015 (link); Lutz et al., 1999 (link)). Briefly, bone marrow cells isolated from the femurs and tibia of 7–14-week-old WT mice were filtered through a 70-μm nylon strainer, red blood cells lysed by ACK lysis buffer (Sigma Aldrich), and cultured in BMDC medium (RPMI-1640 containing 10% FBS, 100 U/mL PS, 2 mM l-glutamine (Gibco), 50 μM 2-mercaptoethanol (Sigma Aldrich), 20 ng/mL GM-CSF (PeproTech), and 10 ng/mL IL-4 (PeproTech)). The BMDC medium was replaced on days 3 and 6. On day 8, non-adherent cells were harvested and BMDC purity was assessed by flow cytometry to ensure staining for markers CD11c, MHC II, CD11b, and CD86. For DC vaccination, non-adherent cells were pulsed for 3 h at 37°C with 10 μM of the human gp100_25–33 (hgp100) peptide (GenScript) in Opti-MEM media (Gibco) and washed three times with PBS before their use. For induction of vitiligo in mice, 1.0×10⁶ CD11c⁺MHC II⁺ hgp100 pulsed BMDCs were co-injected intravenously into WT, IFNaR-deficient or IFNGR-deficient hosts.

Riding R.L., Richmond J.M., Fukuda K, & Harris J.E. (2020). Type I interferon signaling limits viral vector priming of CD8+ T cells during initiation of vitiligo and melanoma immunotherapy. Pigment cell & melanoma research, 34(4), 683-695.

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Isolation and Separation of CNS and Peripheral Immune Cells

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EAE mice were sacrificed by pentobarbital overdose or CO₂ inhalation and transcardially perfused with ice‐cold PBS. Lymph node (LN) and spleen cell suspensions were obtained by mechanical dissociation of the organs and filtering through 40 μm cell strainers in ice‐cold PBS. Erythrocytes were lysed using ice‐cold ACK Lysis buffer (Sigma). Cells were counted using a Scepter Cell Counter (Millipore) and cell discrimination was 4.8–15 µm.
For CNS myeloid cell subset analysis, CNS tissue was minced and digested for thirty min at 37°C in Hank's balanced salt solution containing 50 µg/mL DNaseI (Roche) and 100 µg/mL collagenase/dispase (Roche). The digestion was quenched by cooling samples on ice, passed through a 100 μm mesh, pelleted, resuspended in 30% Percoll (GE Healthcare) and centrifuged at 15,000g for 30 min at 4°C. The myelin layer was removed and the mononuclear cells accumulated in the intermediate phase were collected and further processed.
For T cell cytokine analysis, CNS cells were prepared by homogenization of brains and spinal cords in 50% Percoll solution using a dounce homogenizer. This solution was underlain with 63% Percoll and overlain with 30% Percoll solution before centrifugation at 1000g for 30 min. Cells were then collected at the 30/50% interphase, filtered through a 40 μm cell strained and washed in ice‐cold PBS.

Parsa R., Lund H., Tosevski I., Zhang X., Malipiero U., Beckervordersandforth J., Merkler D., Prinz M., Gyllenberg A., James T., Warnecke A., Hillert J., Alfredsson L., Kockum I., Olsson T., Fontana A., Suter T, & Harris R.A. (2016). TGFβ regulates persistent neuroinflammation by controlling Th1 polarization and ROS production via monocyte‐derived dendritic cells. Glia, 64(11), 1925-1937.

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Isolation and Culture of Lung and Splenic Immune Cells

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Mouse lung or spleen single cell suspension was prepared by mincing whole organs through 40 μm cell strainer (BD Falcon; Franklin Lakes, NJ) followed by red blood cell lysis (ACK lysis buffer) (Sigma-Aldrich; St. Louis, MO) for 3 min. Lung antigen presenting cells were isolated from RBC-free whole lung cells labeled with paramagnetic bead-conjugated anti-CD11c (Miltenyi Biotec; San Diego, CA) and separated using autoMACS (Miltenyi Biotec; San Diego, CA) according to manufacturer's instructions. Mouse Splenic CD4⁺ T cells were isolated from RBC-free single cells suspension from wild type naïve mice as described above; and lung CD11c⁺ mDCs were isolated from RBC-free single cells suspension from WT, C3^−/− and C3ar^−/− mice exposed to air or cigarette smoke. Mouse Splenic CD4⁺ T cells were cultured in complete media for 3 days in vitro with congenic CD11c⁺ lung mDCs (10:1 ratio, 2×10⁵ CD4⁺ T cells and 2×10⁴ mDCs) in the presence of 1μg/ml soluble anti-mouse CD3 (BD; Franklin Lakes, NJ). Milliplex kit (EMD Millipore; Billerica, MA) was used to measure concentrations of a selected group of cytokines (IL-6, IL-17) according to manufacturer's instructions.

Yuan X., Shan M., You R., Frazier M.V., Hong M.J., Wetsel R.A., Drouin S., Seryshev A., MD L.Z., Cornwell L., Rossen R.D., Corry D.B, & Kheradmand F. (2014). Activation of C3a receptor is required in cigarette smoke-mediated emphysema. Mucosal immunology, 8(4), 874-885.

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Isolation and Purification of Murine Splenocytes

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Spleen tissue was aseptically extracted from ICR mice (6-week-old males, 20 ± 2 g). Tissues were disaggregated via passage through a 70 μm nylon mesh (Becton-Dickinson, Franklin Lakes, NJ, USA) in RPMI-1640 medium (Life Technologies), and the cells were purified via centrifugation at 450× g for 5 min. Red blood cells (RBCs) were removed with ACK lysis buffer (Sigma-Aldrich). Splenocytes were then washed with phosphate-buffered saline (PBS), centrifuged at 1000× g for 5 min, and finally suspended in RPMI containing 10% FBS, penicillin, and streptomycin sulfate.

Hwang Y.P., Lee G.H., Pham T.H., Kim M.Y., Kim C.Y., Lee S.Y., Han E.H., Choi C.Y., Hwang S.D., Ahn S, & Jeong H.G. (2022). Immune-Enhancing Effect of Submerged Culture of Ceriporia lacerata Mycelia on Cyclophosphamide-Induced Immunosuppressed Mice and the Underlying Mechanisms in Macrophages. International Journal of Molecular Sciences, 23(2), 597.

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Splenocyte Cytokine Profiling upon rS Wuhan Stimulation

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Mouse spleens were harvested, homogenized and treated with ACK lysis buffer (Sigma Cat. No 11814389001) to remove erythrocyte contamination. Splenocytes were cultured at 1 × 10⁶/well in 96-well plates. Cells were stimulated with 2.5 µg/mL of rS^Wuh for 72 h. IL-2, IL-5 and TNF-α in culture supernatants were measured using an 8-plex multiplex immunoassay (Biolegend Cat No. 741053) according to the manufacturer’s instructions. Data were acquired using a BD FACSCanto™ II, and analysis was performed using Legendplex software (Biolegend, San Diego, CA, USA).

Katsande P.M., Fernández-Bastit L., Ferreira W.T., Vergara-Alert J., Hess M., Lloyd-Jones K., Hong H.A., Segales J, & Cutting S.M. (2022). Heterologous Systemic Prime–Intranasal Boosting Using a Spore SARS-CoV-2 Vaccine Confers Mucosal Immunity and Cross-Reactive Antibodies in Mice as well as Protection in Hamsters. Vaccines, 10(11), 1900.

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Spleen and lymph node dissociation

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Spleen and mLN were harvested, chopped into fine pieces, and digested using collagenase D (1 mg/mL, Sigma, USA) in complete RPMI, for 25 min at 37°C. Cells were filtered and washed with FACS buffer. RBC lysis was carried out for spleen cells using ACK lysis buffer (Sigma, USA).

Nair R., Lannagan T.R., Jackstadt R., Andrusaite A., Cole J., Boyne C., Nibbs R.J., Sansom O.J, & Milling S. (2024). Co-inhibition of TGF-β and PD-L1 pathways in a metastatic colorectal cancer mouse model triggers interferon responses, innate cells and T cells, alongside metabolic changes and tumor resistance. Oncoimmunology, 13(1), 2330194.

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Cytokine Secretion Assay for Immune Cell Activation

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Spleens and lymph nodes were immediately excised from euthanized C57BL/6 or OT-I mice and dissociated in vitro to achieve single-cell suspensions. Red blood cells were lysed with ACK lysis buffer (Sigma-Aldrich) for 2 minutes. Cells were resus-pended at 1 × 10⁶ cells/mL in Iscove's Modified Dulbecco's Medium (IMDM; Gibco) supplemented with 5% FBS, 1% penicillin-streptomycin, 40 μmol/L 2-BME. Cells were cocultured with target B16 or B16MRD cells as described in the text. Cell-free supernatants were then collected 72 hours later and tested for IFNγ (Mouse IFNγ ELISA Kit; OptEIA, BD Biosciences) and TNFα (BD Biosciences) production by ELISA as directed in the manufacturer's instructions.
NK cells were prepared from spleens of naïfive C57BL/6 mice using the NK Cell Isolation Kit II (Miltenyi Biotec) as described in the "NK cell isolation" section and cocultured with B16 or B16MRD target tumor cells at E:T ratios of 20:1. Seventy-two hours later, supernatants were assayed for IFNγ or IL6 by ELISA.

Kottke T., Evgin L., Shim K.G., Rommelfanger D., Boisgerault N., Zaidi S., Diaz R.M., Thompson J., Ilett E., Coffey M., Selby P., Pandha H., Harrington K., Melcher A, & Vile R. (2017). Subversion of NK-cell and TNFα Immune Surveillance Drives Tumor Recurrence. Cancer immunology research, 5(11), 1029-1045.

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