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Pharm lyse lysing solution

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BD Pharm Lyse™ is a lysing solution designed for use in hematological applications. It is a reagent used to lyse or break down red blood cells in order to facilitate the analysis of other blood components, such as white blood cells and platelets.

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Lab products found in correlation

Lsrfortessa, by BD (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) Lymphoprep, by STEMCELL (2 mentions) Tudor dissociation reagent, by BD (2 mentions) Rhapsody scrna seq platform, by BD (2 mentions) Calcein am, by Thermo Fisher Scientific (2 mentions) 70 μm cell strainer, by Corning (2 mentions) Draq7, by BD (2 mentions) Heparin, by Thermo Fisher Scientific (2 mentions) Lineage cell depletion kit, by Miltenyi Biotec (2 mentions) Minimacs, by Miltenyi Biotec (2 mentions) Lung dissociation kit, by Miltenyi Biotec (2 mentions) Gentlemacs dissociator, by Miltenyi Biotec (2 mentions) Collagenase a, by Roche (2 mentions) Rat anti mouse s1p1 apc conjugated antibody, by R&D Systems (2 mentions) Mouse anti human s1p1 eflour 660 conjugated antibody, by Thermo Fisher Scientific (2 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)

40 protocols using pharm lyse lysing solution

1

Isolation of Immune Cell Populations

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Bone marrow cells were isolated as previously described (24 (link)). Briefly, the femurs of age- and sex-matched Nr4a1-deficient and -proficient mice were dissected and flushed with a 27 3/4G needle through a 70 μm nylon strainer and washed two times with PBS.
To obtain splenocytes, freshly isolated spleens were mashed through a 40 µm cell strainer with the plunger of a 1 ml syringe. Subsequently, the cell pellet was resuspended in 3 ml of ammonium-chloride-potassium lysis buffer (155 mM ammonium chloride; 19 mM potassium hydrogen carbonate and 0.68 mM EDTA; pH 7.27) and gently shaken for 3 minutes to lyse erythrocytes.
Lamina propria mononuclear cells (LPMCs) were isolated from the colon using the Lamina Propria Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions followed by Percoll density gradient centrifugation (GE Healthcare).
Peripheral blood was taken from the facial vein. In order to remove erythrocytes, 2 ml of 1x BD Pharm Lyse™ lysing solution (BD Bioscience) was added to 100 µl of whole blood and incubated for 15 minutes at room temperature.
Heidbreder K., Sommer K., Wiendl M., Müller T.M., Atreya I., Hildner K., Neurath M.F, & Zundler S. (2023). Nr4a1-dependent non-classical monocytes are important for macrophage-mediated wound healing in the large intestine. Frontiers in Immunology, 13, 1040775.
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2

Isolation of Blood and Spleen Cells

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Peripheral blood was collected from the facial vein. For erythrocyte removal, 2 mL of 1x BD Pharm Lyse™ lysing solution (BD Bioscience, Franklin Lakes, NJ, USA) was added to 70–80 μL of whole blood, which was vortexed and incubated for 15 min at room temperature (RT). Cells were washed two times with the FACS buffer (phosphate-buffered saline (PBS) supplemented with 1% fetal calf serum (FCS, PAN-Biotech, Aidenbach, Germany), and 2 mM EDTA) and were further processed for flow cytometry.
Splenocytes were isolated as previously described [35 (link)]. In short, freshly isolated spleens were mashed through a 40 μm cell strainer and resuspended in 3 mL of an ammonium–chloride–potassium lysis buffer (155 mM ammonium chloride; 19 mM potassium hydrogen carbonate; 0.68 mM EDTA; and pH 7.27). After 3 min, the cells were washed with PBS and counted with a Neubauer counting chamber. For flow cytometry analysis, 1–2 million splenocytes per sample were used.
Sommer K., Garibagaoglu H., Paap E.M., Wiendl M., Müller T.M., Atreya I., Krönke G., Neurath M.F, & Zundler S. (2024). Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies. Cells, 13(10), 819.
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3

Enrichment of Circulating Epithelial Tumor Cells

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We collected 2.5 ml of peripheral blood samples from ESCC patients and healthy volunteers in EDTA tubes. Density gradient centrifugation was performed using the RosetteSep™ Human Circulating Epithelial Tumor Cell Enrichment Cocktail (StemCell™ Technologies, Vancouver, Canada) combined with Lymphoprep™ (StemCell™ Technologies, Vancouver, Canada). To the 2.5 ml blood sample was added 250 µl (50 µl/ml) of the RosetteSep™ cocktail and then incubated for 20 min at room temperature. Blood samples were diluted with equal volumes of PBS and carefully layered onto Lymphoprep™ then centrifuged at 3,600 rpm at room temperature for 20 min. After centrifugation, supernatants were transferred to another 15 ml conical tube with cells pelleted by centrifugation at 1,800 rpm for 20 min at room temperature. The enriched cells were collected, red blood cells were lysed by BD Pharm Lyse lysing solution (Becton, Dickinson and Company, New Jersey, USA), and washed in PBS.
Akashi T., Okumura T., Terabayashi K., Yoshino Y., Tanaka H., Yamazaki T., Numata Y., Fukuda T., Manabe T., Baba H., Miwa T., Watanabe T., Hirano K., Igarashi T., Sekine S., Hashimoto I., Shibuya K., Hojo S., Yoshioka I., Matsui K., Yamada A., Sasaki T, & Fujii T. (2023). The use of an artificial intelligence algorithm for circulating tumor cell detection in patients with esophageal cancer. Oncology Letters, 26(1), 320.
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4

Apoptosis and Oxidative Stress in VAP

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Blood was collected from patients with proved VAP, who had being received mechanical ventilation for 72 hours and longer. Specimens were received for the first time before the beginning of amikacin inhalation (main observation group) or correction of systemic antibiotic management of VAP according to the conventional protocols (comparison group), following 48 h and 96 h after the beginning of antimicrobial management of VAP in both groups of patients, respectively. For blood samples we used plastic tubes containing EDTA di-potassium salt (IMPROVACUTER, Guangzhou, China).
The freshly collected samples were used to obtain erythrocyte and leukocyte suspensions. To obtain erythrocyte suspensions, blood samples were washed twice with phosphate-buffered saline (PBS, pH 7.4) purchased from Becton Dickinson (USA). Thereafter, 2 µL of red blood cell mass was dissolved in 100 µL BD PharmingenTM Annexin V Binding Buffer (BD Biosciences, USA) for determination of phosphatidylserine externalization or PBS for ROS detection.
To obtain leukocyte suspensions, blood aliquots of 100 µL were incubated for 15 min with 2 ml BD Pharm Lyse™ lysing solution purchased for Becton Dickinson (USA). After incubation, the suspensions were washed twice with PBS and resuspended in 100 µL annexin-binding buffer for Annexin V/7-aminoactinomycin D (7AAD) staining or PBS for H2DCFDA staining.
Levchenko B., Nazarchuk O., Dmytriiev D., Bahniuk N., Melnychenko M, & Dmytriiev K. (2023). Adjunctive inhaled amikacin in infants with ventilator-associated pneumonia optimizes the complex antimicrobial therapy: pilot study. Acta Bio Medica : Atenei Parmensis, 94(2), e2023084.
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5

Extracellular Acidification Profiling of CTCs and WBCs

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To identify a threshold of extracellular acidification rate (i.e., pH) able to better discriminate between CTCs and WBCs, the ECAR of breast cancer cell lines (MDA-MB-231 and MCF7), and WBCs was separately assessed by using the MBA. WBCs were obtained from the peripheral blood of healthy donors (n = 3), after red blood cell lysis with BD Pharm Lyse lysing solution (Beckton-Dickinson, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions, while breast cancer cell line (MCF7 and MDA-MB-231), grown as previously described at a confluence of 70–80%, were detached with 1% Trypsin-EDTA. Cells were resuspended at a concentration of 1,000,000 cells/mL in 50 μL of the unbuffered Joklik’s modified EMEM culture medium (Sigma) containing 2 mM EDTA, 0.1% BSA, 15% Optiprep and 4 mM of the fluorescent pH indicator SNARF-5F, emulsified as described in the previous section, incubated at 37 °C for 30 min and analyzed. All experiments were performed in triplicate at various cell passages.
Brisotto G., Biscontin E., Rossi E., Bulfoni M., Piruska A., Spazzapan S., Poggiana C., Vidotto R., Steffan A., Colombatti A., Huck W.T., Cesselli D., Zamarchi R., Turetta M, & Del Ben F. (2020). Dysmetabolic Circulating Tumor Cells Are Prognostic in Metastatic Breast Cancer. Cancers, 12(4), 1005.
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6

Neuronal Cell Isolation and Staining

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Trypsin (27252-018), collagenase II (QN0512), and DMEM (12800-017) were purchased from GIBCO. Fetal bovine serum (04-001-1ACS) was purchased from Biological Industries. Polylysine (P0296) was purchased from Sigma. DAPI (ab104139), anti-NeuN (ab177467), and Goat anti-rabbit IgG H&L (FITC, ab6717) were purchased from Abcam. BD Pharm Lyse lysing solution (No. 555899) was purchased from Becton Dickinson. The stationary liquid 0.01 M PBS solution) contained 3% formaldehyde and 20% sucrose. BD FACS Celesta Flow cytometry was obtained from BD Biosciences-US, and Cytation5 was obtained from BioTek.
Li J., Zhang Y., Zhang T., MeiYuan T., Hou J., Huang D., Cheng Y.Z., Zhu M., Su X., Li Z., TONG S., Zhang X., Deng J., DONG Y., & Ma Y. (2020). Analysis of isolation of cerebral cortical neurons in rats by different methods. Biocell, 44(2), 209-215.
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7

Isolation of PBMCs from Blood

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Peripheral blood mononuclear cells (PBMCs) were obtained from 2ml EDTA-k2 treated blood sample with 1 × BD Pharm Lyse lysing solution (BD Pharmingen, USA) according to the instructions from the manufacturer. PBMCs were washed twice in 10ml phosphate buffered saline (PBS) (Corning, Manassas, VA, USA) with 1% fetal bovine serum (FBS) (Gibco, Life Technologies, USA) and collected by centrifugation at the speed of 300× g for 5 min.
Zhao J., Lin B., Deng H., Zhi X., Li Y., Liu Y., Bible P.W., Li Q., Xu B., Wei L., Yang H, & Huang D. (2018). Decreased Expression of TIM-3 on Th17 Cells Associated with Ophthalmopathy in Patients with Graves’ Disease. Current Molecular Medicine, 18(2), 83-90.
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8

Quantifying Monocyte Surface Markers

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Blood was collected by cardiac puncture and the red blood cells removed with Pharm Lyse lysing solution (BD). Leukocytes were incubated at 37°C for 1 h with 20 μg/ml of either mStab1–1.26- or MOPC-21-Alexa Fluor 647 (conjugated as described above), washed, stained with α-CD11b-PE and α-Ly6C-BV421 and analyzed by flow cytometry. The geometric mean fluorescence intensities of the Ly6Chigh and Ly6Clow monocyte populations of wildtype mice were normalized to the unspecific antibody binding of the corresponding populations of Stab1−/− mice, which were set at 100%.
Dunkel J., Viitala M., Karikoski M., Rantakari P., Virtakoivu R., Elima K., Hollmén M., Jalkanen S, & Salmi M. (2018). Enhanced Antibody Production in Clever-1/Stabilin-1–Deficient Mice. Frontiers in Immunology, 9, 2257.
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9

Dissociation and Single-Cell Preparation of NSCLC Tumors

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Surgically resected NSCLC tumor tissues and adjacent normal tissues were minced into small pieces (<1 mm) on ice and enzymatically digested with agitation for 30 min at 37°C using the BD TuDoR™ dissociation reagent (BD Biosciences). The obtained single-cell solution was sieved through a 70 μM cell strainer (Corning) and red blood cells were removed using the BD Pharm Lyse™ lysing solution (BD Biosciences). Cells were counted and viability assessed with the BD Rhapsody scRNA-seq platform (BD Biosciences) using Calcein-AM (Invitrogen) and Draq7 (BD Biosciences).
Salcher S., Sturm G., Horvath L., Untergasser G., Kuempers C., Fotakis G., Panizzolo E., Martowicz A., Trebo M., Pall G., Gamerith G., Sykora M., Augustin F., Schmitz K., Finotello F., Rieder D., Perner S., Sopper S., Wolf D., Pircher A, & Trajanoski Z. (2022). High-resolution single-cell atlas reveals diversity and plasticity of tissue-resident neutrophils in non-small cell lung cancer. Cancer Cell, 40(12), 1503-1520.e8.
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10

Isolation of Bone Marrow Stem Cells

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Bone marrow (BM) was collected from ALS patients after obtaining their informed consent. Each time, 40-50 mL of BM samples were harvested in local anesthesia from the posterior iliac crest and subsequently resuspended in collecting medium (PBS, pH 7.2) and heparin (20 U/mL; Gibco, USA). The collected BM was lysed in BD PharmLyse Lysing Solution (BD Biosciences, San Jose, USA) for 15 min at room temperature in the dark and washed twice in phosphate-buffered saline (PBS). The procedure was performed in compliance with the manufacturer's protocol, as previously described 11 . The obtained suspension of BM nucleated cells (NCs) was subjected to immunomagnetic separation (MiniMACS, Miltenyi Biotec, Auburn, USA). LIN- stem/progenitor cells (mean 8.98 ± 5.77×106) were isolated from non-separated NCs using immunomagnetic isolation and a Lineage Cell Depletion Kit (Miltenyi Biotec, Auburn, USA), as previously described 20 (link). Before administration, the isolated cells were kept in 2 mL of sterile PBS.
Pawlukowska W., Baumert B., Gołąb-Janowska M., Pius-Sadowska E., Litwińska Z., Kotowski M., Meller A., Rotter I., Peregud-Pogorzelski J, & Nowacki P. (2020). Articulation recovery in ALS patients after lineage-negative adjuvant cell therapy - preliminary report. International Journal of Medical Sciences, 17(13), 1927-1935.
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