After euthanasia, BALF samples were obtained by centrifuging at 1800 rpm for 3 min with sterile normal saline (0.9% NaCl). To assess immune cells in alveoli, pellets from BALF centrifugation were mixed for total cell counts with a Z1 particle counter (Beckman-Coulter, Indianapolis, IN, USA). Cytospin slides were prepared using cells in the BALF and stained with Diff-Quick (Baxter, Chicago, IL, USA). Under light microscopy, immune cell populations, including neutrophils, eosinophils, basophils, macrophages, and lymphocytes, were counted.
Diff quick
Manufactured by Baxter
Sourced in United States, United Kingdom
Diff-Quick is a rapid staining kit used for the differential staining of blood smears and other cellular samples. It consists of three solutions that sequentially stain the cellular components, allowing for the identification and differentiation of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
48 well boyden microchemotaxis chamber, by Neuro Probe (4 mentions)
Zap oglobin 2, by Beckman Coulter (3 mentions)
Mba 96, by Neuro Probe (2 mentions)
Model z1, by Beckman Coulter (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Z1 particle counter, by Beckman Coulter (1 mentions)
Hemocytometer, by Hausser Scientific (1 mentions)
Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Z2 particle counter and size analyzer, by Beckman Coulter (1 mentions)
Hemavet 950 analyzer, by Drew Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by BD (1 mentions)
Forane, by Baxter (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Elisa immunoassay kit, by R&D Systems (1 mentions)
48 well microchemotaxis chamber, by Neuro Probe (1 mentions)
Pneumocystis murina, by American Type Culture Collection (1 mentions)
Vi cell, by Beckman Coulter (1 mentions)
Matched antibody pairs, by R&D Systems (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
29 protocols using diff quick
1
Analyzing Alveolar Immune Cells in BALF
2
Automated Blood Cell Analysis and Cytokine Quantification
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A 50-μL aliquot of blood was used for automated CBC analysis (Hemavet, Drew Scientific, Miami, Fla). The remainder of the blood was centrifuged (2,000 × g, 5 min) and the plasma stored at −20°C for later cytokine analysis. The peritoneal lavage fluid was centrifuged (600 × g, 5 min), and the supernatant saved at −20°C for later cytokine analysis. The cell pellet was resuspended in 200 μL RPMI 1640 (Invitrogen) containing 0.1% heat-inactivated fetal bovine serum (Invitrogen). Cells were counted after RBC lysis (Zap-O-globin II (Beckman Coulter, Indianapolis, Ind)) using a hemocytometer (Hausser Scientific, Horsham, Pa). Experimental groups were known to the operator during initial cell counts but evaluation of differentials was blinded. Slides were loaded with 1 × 105 cells, centrifuged (109 × g, 5 min), and stained with Diff-Quick (Baxter, Detroit, Mich). Differentials (300 cells) were counted under light microscopy.
3
Differentiation and Infection of Murine Bone Marrow-Derived Dendritic Cells
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Bone marrow from the femurs and tibias of C57Bl/6 mice was collected and cultured with complete RPMI medium supplemented with 10% (v/v) FBS and gentamicin (20 µg/ml). Recombinant IL-4 and GM-CSF (20 ng/ml) were used to differentiate DCs for 7–8 days to obtain 80–90% purity of CD11c+ DCs that was verified by flow cytometry. BMDCs were harvested and plated on 24-well tissue culture plates. For early infection studies and measurements of CD200 expression and cytokines, DCs were infected with parasites at a ratio of 1:10 (DC:parasite) for 6 h at 37°C. Thereafter, DCs were thoroughly washed with PBS to remove non-internalized parasites. The number of amastigotes in infected DCs was stained with Diff-Quick (Baxter Healthcare Corporation) and parasite number was evaluated microscopically. The expression of CD200, number of intracellular amastigotes, and NO production were measured at 1, 4, 24, and 48 h post incubation of infected DCs following previously published protocols (11 (link)). Cytokines released by infected DCs were measured in culture supernatants collected at 24, 48, and 72 h post infection.
4
Boyden Chemotaxis Assay for BMDM
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Chemotaxis of BMDM was performed using a modified Boyden chemotaxis chamber containing a 96-well microchemotaxis plate (MBA-96; Neuro Probe, Cabin John, MD) as described previously with minor modifications [70 (link)]. Briefly, the bottom wells of the chamber contained 40 μl of HA6 (4 mM) dissolved in defined medium (DM), without FCS. Positive and negative controls included 10% FCS or DM, respectively. A 5-μm-pore polycarbonate membrane filter was placed between the bottom and top chamber. The upper wells were filled with 1 × 106 cells/ml suspended in 10 μl DM. The chamber was incubated for 6 h at 37 °C. Non-migratory cells on the upper surface of the membrane were treated with 200 μl of 1 mM EDTA for 15–20 min and wiped off. Cells that had migrated into the membrane were stained with Diff-Quick (Baxter Healthcare, McGaw Park, IL) and counted in five randomly selected high-power fields in each well. Each chemoattractant solution was tested in six wells and each experiment was repeated at least three times. Data were expressed as the number of macrophages that migrated into the membrane for each condition and converted to a percentage of control (DM) and data from three separate experiments were combined.
5
Cell Differential Analysis from BAL and PL Fluids
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The BAL and PL fluids were centrifuged (600g, 5 min). Supernatants were stored at −20 °C. The pellets from the two samples were pooled, red blood cells lysed with Zap-Oglobin II® (Coulter Corp. Miami, FL, USA), and total cell counts performed with a Coulter Counter model Z1. Slides were loaded with 1 × 105 cells, centrifuged (700g, 3 min) and stained with Diff-Quick (Baxter, Detroit, MI, USA). Differentials (300 cells) were counted and used to calculate absolute cell counts.
6
Bronchoalveolar Lavage Fluid Analysis
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A tracheotomy was performed in the sacrificed mice. After insertion of the tracheal tube, the whole lung was lavaged two times with 1-ml sterile saline at room temperature. The recovered fluids were filtered through a single layer of gauze to remove mucus. Cells in the BALF were counted using a hemocytometer. Differential counts of BAL cells were performed on 200 cells stained with Diff-Quick (Baxter Diagnostics, Düdingen, Switzerland).
7
Bronchoalveolar Lavage Fluid Collection
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Trachea was exposed through midline incision and intubated with aseptic 22 Abbocath-T catheter (Abbott, Sligo, Ireland). BAL was performed by injecting two parts of 0.5 mL aseptic saline into the right lung. The recovered BALF (0.8 mL) was rotated at 4 °C for 10 min at 260×g, and the precipitate was re-suspended in 0.5 mL sterile PBS. The total number of cells was counted by a Z2 Particle Counterand Size Analyzer (Beckman-Coulter, Miami, FL, USA). Differential cell counting (Diff-Quick, Baxter, UK) was performed on cell centrifuge smears stained with modified Giemsa stain.
8
Unilateral Bronchoalveolar Lavage Procedure
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BALF was obtained as previously described [23] . The trachea and bronchi were exposed through a midline incision. The left main bronchus was ligated and the trachea was cannulated with a sterile 22-gauge Abbocath-T catheter (Abbott Laboratories, Sligo, Ireland). Unilateral, right-sided BAL was performed by instilling three 0.3 mL aliquots of sterile PBS. 0.7-0.9 mL of BALF was retrieved per mouse. Total cell numbers in BALF were counted using a Z2 Coulter particle count and size analyser (Beckman-Coulter, Inc., Miami, FL, USA). BALF differential cell counts were carried out on cytospin preparations stained with modified Giemsa stain (Diff-Quick; Baxter, McGraw Park, IL, USA).
9
CLP-Induced Sepsis: Fibrocyte Transfer
10
Boyden Chamber Assay for Cell Migration
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Cell migration assay was performed using a 48-well Boyden microchemotaxis chamber (Neuro Probe Inc., Gaithersburg, MD, USA), as in a previous report [16 (link)]. Briefly, lower chamber wells were filled with DMEM containing 0.1% BSA and EGF (1 ng/mL) or different concentrations of ITMFAb. A membrane (Neuro Probe, Cabin John, MD, USA) coated with type Ι collagen (0.1 mg/mL) was then laid over lower chamber wells, and upper chamber wells were loaded with HaCaT cells (5 × 104 cells/well) in DMEM containing 0.1% BSA. Chambers were assembled and incubated for 210 min at 37 °C. Membranes were then removed, fixed, and stained using Diff-Quick (Baxter Healthcare, Miami, FL, USA). The cells migrating to lower membrane surfaces were counted using an optical microscope (Carl Zeiss, Jena, Jena, Thuringen, Germany) (×200).
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