Ack cell lysing buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

ACK cell lysing buffer is a solution used to lyse red blood cells (erythrocytes) in biological samples, typically to prepare samples for further analysis. The buffer contains ammonium chloride, potassium bicarbonate, and EDTA, which work together to effectively and gently lyse red blood cells while preserving the integrity of other cell types, such as white blood cells or other target cells of interest.

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Lab products found in correlation

Liberase, by Roche (2 mentions) Dnase 1, by Roche (2 mentions) Navios, by Beckman Coulter (1 mentions) Anti biotin microbead, by Miltenyi Biotec (1 mentions) F4 80 bm8, by Thermo Fisher Scientific (1 mentions) Cd27 lg 3a10, by BioLegend (1 mentions) Ter 119 ter119, by Thermo Fisher Scientific (1 mentions) Cd11c n418, by Thermo Fisher Scientific (1 mentions) Cd49b dx5, by Thermo Fisher Scientific (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions)

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ACK lysing buffer (559 citations) ACK buffer (154 citations) Lysing buffer (12 citations)

5 protocols using ack cell lysing buffer

Murine Bone Marrow-Derived Dendritic Cells

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DCs precursors were harvested from murine bone marrow (BM). Briefly, BM from the tibiae and femurs of 6- to 8-week-old male C57BL/6 mice were flushed with RPMI and depleted of red blood cells with ACK cell lysing buffer (GIBCO, Life Technologies, Walthan, MA, USA). Cells were plated in 6-well culture plates (1 × 10⁶ cells/mL; 3 mL/well) in RPMI supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 25 μg/mL rmGM-CSF, and 25μg/mL rmIL-4 at 37 °C in a humidified 5% CO₂ atmosphere. On day 3, BMDCs were harvested and plated at 1 × 10⁶/mL in 24-well culture plates. DCs were cultured from murine bone marrow as previously described [37 (link)]. On day 7 BMDCs were treated with MTE raw extract and MTE_mp. Lipopolysaccharide (LPS) was administered (1 μg/mL) at day 8 and 24 h later the supernatant (SN) was harvested.
Cytofluorimetric analysis. Cells were stained with Anti-MHC Class II antibodies (clone: M5/114.15.2) and Anti-CD11c-FITC (clone: N418) (Miltenyi Biotec, Bergisch Gladbach, Germany) following manufactures protocol. Flow Cytometer acquisition was performed using NAVIOS (Beckman Coulter, Brea, CA, USA).

Sansone F., Esposito T., Lauro M.R., Picerno P., Mencherini T., Gasparri F., De Santis S., Chieppa M., Cirillo C, & Aquino R.P. (2018). Application of Spray Drying Particle Engineering to a High-Functionality/Low-Solubility Milk Thistle Extract: Powders Production and Characterization. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(7), 1716.

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Dendritic Cell Differentiation and Stimulation

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Dendritic cells were harvested from murine bone marrow. Briefly, bone marrows from the tibiae and femurs of 6- to 8-week-old male C57Bl/6 mice were flushed with RPMI and depleted of red blood cells with ACK cell lysing buffer (GIBCO). The cells were plated in 6-well culture plates (1Å–10⁶ cells/mL; 3 mL/well) in RPMI supplemented with 10% heat-inactivated FBS, 100-U·mL⁻¹ penicillin, 100-mg·mL⁻¹ streptomycin, 25-µg·mL⁻¹ rmGM-CSF, and 25-µg·mL⁻¹ rmIL-4 at 37°C in a humidified 5% CO2 atmosphere. On day 3, bone marrow DCs (BMDCs) were harvested and plated at 1 × 10⁶ mL⁻¹ on 24-well culture plates. On day 7, BMDCs were administered with tomato methanol extracts (0.1-g lyophilized powder·mL⁻¹) at a 1:25 final dilution. Lipopolysaccharide (LPS) was administered [1 µg·mL⁻¹] at day 8 for 24 h. DC viability was assessed by cytofluorimetric analysis (Supplementary Methods in Supplementary Material).

Scarano A., Butelli E., De Santis S., Cavalcanti E., Hill L., De Angelis M., Giovinazzo G., Chieppa M., Martin C, & Santino A. (2018). Combined Dietary Anthocyanins, Flavonols, and Stilbenoids Alleviate Inflammatory Bowel Disease Symptoms in Mice. Frontiers in Nutrition, 4, 75.

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Murine Dendritic Cell Generation and Polyphenol Treatment

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DCs were harvested from murine bone marrow (BM). Briefly, BM from the tibiae and femurs of 6- to 8-week-old male C57BL/6 mice were flushed with RPMI and depleted of red blood cells with ACK cell lysing buffer (GIBCO). Cells were plated in 6-well culture plates (1×10⁶ cells/ml; 3 ml/well) in RPMI supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 25 µg/ml rmGM-CSF, and 25 µg/ml rmIL-4 at 37°C in a humidified 5% CO₂ atmosphere. On day 3, BMDCs were harvested and plated at 1×10⁶/ml in 24-well culture plates. On day 5 and day 7, BMDCs were administered polyphenols (25 µM). LPS was administered [1 µg/ml] at day 8 for 24 h.

Cavalcanti E., Vadrucci E., Delvecchio F.R., Addabbo F., Bettini S., Liou R., Monsurrò V., Huang A.Y., Pizarro T.T., Santino A, & Chieppa M. (2014). Administration of Reconstituted Polyphenol Oil Bodies Efficiently Suppresses Dendritic Cell Inflammatory Pathways and Acute Intestinal Inflammation. PLoS ONE, 9(2), e88898.

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Isolation and Enrichment of Innate Lymphoid Cells

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Lungs were perfused with PBS, diced into ~2-cm pieces and incubated with Liberase TM (42.4 μg/ml) and DNAse I (10 U/ml; both from Roche) for 45 min at 37 °C before being mashed through a 70-μM cell strainer and washed with complete RPMI. Remaining blood cells were lysed with ACK cell lysing buffer (Invitrogen) and single cell suspensions were incubated with biotinylated antibodies to CD3ε (clone 145-2C11), CD19 (1D3), B220 (RA3-6B2), CD5 (53-7.3), TCRβ (H57-597), TCRγδ (GL3), CD11c (N418), F4/80 (BM8), Gr-1 (RB6-8C5), Ter119 (TER-119), CD49b (DX5; all eBioscience) and CD27 (LG3A10, BioLegend) all at 25 μg/ml per 2 × 10⁸ cells. Cells were then incubated with anti-biotin microbeads (Miltenyi; 1:5 dilution at 10⁸ cells per ml) and depleted following manufacturer’s protocol. For each ILC enrichment, the depletion was repeated twice and yielded >95% pure ILC populations (Supplementary Fig. 3a).

Silver J.S., Kearley J., Copenhaver A.M., Sanden C., Mori M., Yu L., Pritchard G.H., Berlin A.A., Hunter C.A., Bowler R., Erjefalt J.S., Kolbeck R, & Humbles A.A. (2016). Inflammatory triggers associated with exacerbations of COPD orchestrate plasticity of group 2 innate lymphoid cells in the lungs. Nature immunology, 17(6), 626-635.

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Lung Tissue Collection and Processing

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Mice were anesthetized by a subcutaneous injection of 0.25 mL of (v:v) ketamine 50 mg mL⁻¹ and xylazine 20 mg mL⁻¹ (Centravet, Nancy, France) and exsanguinated via the inferior vena cava. The right upper and lower lung lobes were resected and homogenized in 1 mL Tris/EDTA/Tween buffer containing protease inhibitors and TriZol (Life Technologies, Saint Aubin, France) for protein and total RNA isolation, respectively. The right median and accessory lobes were cryopreserved in liquid nitrogen and stored at −80 °C. The left lung lobe was inflated and fixed in 4% buffered formaldehyde for 24 h at 4 °C and processed for obtaining paraffin-embedded blocks. Five micrometre lung tissue sections were either stained with hematoxylin and eosin for morphological examination, or they were used for immunohistochemistry.
For experiments involving flow cytometry, lungs were perfused with PBS, cut into ~2 mm pieces and incubated with Liberase (42.4 μg mL⁻¹) and DNAseI (10 U mL⁻¹; both from Roche) for 45–60 min at 37 °C before being mashed through a 70 μm cell strainer and washed with complete RPMI. Remaining blood cells were lysed with ACK cell lysing buffer (Invitrogen) and single cell suspensions were obtained for further analysis.

Dagher R., Copenhaver A.M., Besnard V., Berlin A., Hamidi F., Maret M., Wang J., Qu X., Shrestha Y., Wu J., Gautier G., Raja R., Aubier M., Kolbeck R., Humbles A.A, & Pretolani M. (2020). IL-33-ST2 axis regulates myeloid cell differentiation and activation enabling effective club cell regeneration. Nature Communications, 11, 4786.

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