Bone marrow cells were isolated from the femur of ubiquitin-ERT2-Cre Tsc2 fl/fl mice and stained with antibodies for flow sorting. After lysis of erythrocytes in ACK buffer, cells were washed in PBS supplemented with 0.1% BSA and resuspended in complete RPMI. Common myeloid progenitors (CMP, Lin-ckit+CD32loCD34hi), granulocyte-monocyte progenitors (GMP, Lin-ckit + CD32hiCD115lo), common monocyte progenitors (CMoP, Lin-ckit+CD32hi CD115hi Ly6Chi), and Ly6Chi monocytes (ckit-CD115hiLy6Chi) were sorted by a FACSAriaTM Fusion Cell Sorter (BD). Sorted cells were cultured in complete RPMI medium with stem cell factor (40 ng/mL) and 4OH-tamoxifen (1 μM) for 6 days prior to the addition of M-CSF (20 ng/mL). On day 13, whole blood (10 μL) from wild-type mice was added to the cultured cells to induce hemophagocytosis. After 24 h, hemophagocytes were imaged following Giemsa Wright staining (Sigma-Aldrich).
Wright giemsa staining
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Wright-Giemsa staining is a laboratory technique used for staining blood smears and other cellular samples. It is a combination of the Wright stain and the Giemsa stain, which together provide a differential staining of cellular components. The staining process allows for the visualization and identification of various blood cell types, including red blood cells, white blood cells, and platelets. This technique is commonly used in hematology and cytology laboratories for diagnostic purposes.
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Lab products found in correlation
Axiocam ic camera, by Zeiss (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Fsx100 microscope, by Olympus (1 mentions)
Ferric nitrate nonahydrate, by Merck Group (1 mentions)
Human transferrin, by Merck Group (1 mentions)
Aggrewell eb formation medium, by STEMCELL (1 mentions)
Stemline 2, by Merck Group (1 mentions)
Cytospin 4, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Siglec f, by BioLegend (1 mentions)
Ly6g fitc, by BD (1 mentions)
Cd45 pe cy7, by BD (1 mentions)
Apc gr 1, by BD (1 mentions)
F4 80 pe, by Thermo Fisher Scientific (1 mentions)
Cd11b apc cy7, by BD (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
Tunel staining, by Merck Group (1 mentions)
8 protocols using wright giemsa staining
1
Hemophagocytosis Assay of Myeloid Progenitors
2
Giemsa-Wright Staining of Cells
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Giemsa-Wright staining was performed according to the manufacturer’s instructions (Sigma Aldrich). Briefly, 105 cells in 300 μL media were spun onto a Cytospin slide and subsequently fixed with methanol for 15 min. After drying, slides were incubated with 500 µL of Wright solution for 2 min, rinsed with dH2O, dried again, and incubated with a 1:20 Giemsa solution diluted with dH2O for 1 h. Afterwards, the slides were thoroughly washed with dH2O and dried. Imaging was performed using a Zeiss Axiovert200M microscope (Oberkochen, Germany), a Plan-Apochromat 63 × oil objective (Zeiss) and an AxioCam IC camera (Zeiss).
3
Saliva Cell Staining Protocol
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We used Wright-Giemsa staining (Sigma, MO) to visualize cellular components of saliva following the standard protocol. Briefly, around 0.5 ml of saliva was transferred onto a glass slide without bubbles and spread out to a thin, even layer using the edge of another glass slide. After air-drying for several minutes, the slide was stained with 1–2 ml of the Wright-Giemsa stain for two minutes and rinsed with deionized water. Excess water was blotted and the slide was air-dried. Images of stained cells were captured using a brightfield mode on the Olympus FSX-100 microscope (Olympus, PA).
4
Embryoid Body-Derived Erythroid Differentiation
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An embryoid body was induced using AggreWell EB formation medium (StemCell Technologies). Three days after incubation in AggreWell, the embryoid body was placed on a layer of confluent adherent OP9 cells. After microscopically confirming the wheel shape of cells co-cultured for eight days, the cells were collected using collagenase IV and trypsin/EDTA.
On day 1 of differentiation to the erythroid lineage, a single-cell suspension of eight-day-co-cultured cells was prepared in erythroid basal medium containing Stemline II, 150 μg/mL human transferrin, 50 μg/mL insulin, 90 ng/mL ferric nitrate nonahydrate, and 160 μM monothioglycerol solution (all from Sigma-Aldrich, St. Louis, MO, USA). The medium was refreshed once every 2–3 days. Cell morphology was evaluated using cytocentrifugation (Cytospin 4, Thermo Fisher Scientific; 700 rpm for 7 min) and Wright–Giemsa staining (Sigma-Aldrich).
On day 1 of differentiation to the erythroid lineage, a single-cell suspension of eight-day-co-cultured cells was prepared in erythroid basal medium containing Stemline II, 150 μg/mL human transferrin, 50 μg/mL insulin, 90 ng/mL ferric nitrate nonahydrate, and 160 μM monothioglycerol solution (all from Sigma-Aldrich, St. Louis, MO, USA). The medium was refreshed once every 2–3 days. Cell morphology was evaluated using cytocentrifugation (Cytospin 4, Thermo Fisher Scientific; 700 rpm for 7 min) and Wright–Giemsa staining (Sigma-Aldrich).
5
Isolation and Characterization of Alveolar Macrophages
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BALF was collected as described (Li et al, 2013 (link)) and centrifuged at 400g for 10 min to separate the supernatant from cells. The numbers of total cells, neutrophils, and macrophages from BALF were visualized using the Wright–Giemsa staining (Sigma-Aldrich) and counted in a double-blind manner. Isolation of alveolar macrophages from BALF was performed as described in detail earlier (Busch et al, 2019 (link)), and the purity of isolated macrophages was characterized by dual staining with PE-conjugated sialic acid–binding immunoglobulin-like lectin F (Siglec-F) and Alexa Fluor 488–conjugated CD11c (BioLegend) followed by flow cytometry.
6
Isolation and Characterization of Tumor-Infiltrating Myeloid Cells
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Tumors were harvested from MMTV-PyVT transgenic mice or MCaP0008 tumor-bearing mice. Tumor tissues were then minced and digested at 37°C for 45 min with DMEM containing collagenase type 1A (1.5 mg/ml), hyaluronidase (1.5 mg/ml), and DNase (20 U/ml). TiMDSCs and TAMs were enriched by CD11b-microbeads (Miltenyi, Germany). Enriched cells were then stained with PE-F4/80 (eBioscience), FITC-Ly6G, PE-Cy7-CD45, APC-Gr1, APC-Cy7-CD11b (BD Biosciences) and isolated by flow sorting. 50,000 tiMDSCs were subjected to cytospin preparation (Thermo Shandon), and were fixed and stained with a Wright Giemsa Staining according to the manufacturer's instructions (Sigma-Aldrich).
7
Investigating Neutrophil Survival in HSC Co-culture
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HSCs were isolated and cultured in RPMI-1640 medium with 10% FBS and 10% horse serum. One day or 5 days later, the isolated neutrophils were added (1 × 106 cells per well of a 12-well-plate). The live neutrophil number was determined by using Trypan Blue staining 1–4 days later. The neutrophils were analyzed further with Wright-Giemsa staining (Sigma-Aldrich) and TUNEL staining (Millipore, Cleveland, OH). The HSC conditioned medium was prepared 24 hours after the refreshment of the medium of the 5-day cultured HSCs. When indicated, neutralizing antibodies against GM-CSF (cat number: MAB415-100; R&D, Minneapolis, MN) and IL-15 (cat number: AF447-SP; R&D) were applied to the co-culture system.
8
Bronchoalveolar Lavage Fluid Analysis
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BALF was centrifuged for 10 min at 4 °C and 1500 ×g. The precipitated cells were resuspended again in PBS buffer. Cells were observed by Wright-Giemsa staining (Sigma-Aldrich) and counted in a double-blind method. The concentration of protein in the supernatant was measured using the bicinchoninic acid (BCA) protein detection method (Pierce, Rockford, IL, USA).
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