A bronchoalveolar lavage (BAL) was performed following completion of lung function in anesthetized rabbits. A cannula was inserted within the endotracheal tube, and 5 ml of sterile saline was administered into the lung followed by recovery of the fluid under gentle vacuum suction. The fluid was left in the lung for no more than a few seconds, and the amount of fluid recovered was noted (approximately 60% recovery). An aliquot of the lavage fluid (50 μl) was fixed with 50 μl of Turk’s solution (catalog number 109277; Merck KGaA, Darmstadt, Germany). Total cell counts were performed for this solution using a Neubauer hemocytometer. Two 100 μl samples of neat BAL fluid were centrifuged (Cytospin 3 Centrifuge; Thermo Scientific Shandon, Waltham, MA) onto a glass slide. The slides were left to dry, fixed (Reastain Quick-Diff Kit; Reagena, Toivala, Finland), and then mounted in DPX Mountant (Sigma-Aldrich). Differential cell counts were performed using confocal microscopy with oil at a 40× magnification.
Turk s solution
Manufactured by Merck Group
Sourced in Germany
Turk's solution is a laboratory reagent used for the determination of total protein concentration in biological samples. It is a copper-based solution that reacts with peptide bonds in proteins, producing a colored complex that can be measured using a spectrophotometer. The intensity of the color is proportional to the protein concentration in the sample.
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Lab products found in correlation
Dpx mountant, by Merck Group (1 mentions)
Edta tube, by BD (1 mentions)
Mouse fc block, by BD (1 mentions)
Neubauer hemocytometer, by Merck Group (1 mentions)
λ carrageenan, by Merck Group (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Penicillin, by BD (1 mentions)
Rifn γ, by Thermo Fisher Scientific (1 mentions)
Ril 4, by R&D Systems (1 mentions)
Ultraglutamine, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Neubauer chamber, by Merck Group (1 mentions)
F4 80 pe, by BD (1 mentions)
Cd19 pe, by BD (1 mentions)
Cd8 pe cy7, by BD (1 mentions)
Cd11b bv510, by BD (1 mentions)
Abx pentra 60c, by Horiba (1 mentions)
Easylyse, by Agilent Technologies (1 mentions)
Ly6g pe cy7, by BD (1 mentions)
Ly6c fitc, by BD (1 mentions)
11 protocols using turk s solution
1
Rabbit Lung Cell Differential Analysis
2
Quantifying Ovine Colostral PMNs
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To determine the quantity of ovine colostral PMN, cells from 15 healthy Merino sheep ewes (n = 15) were manually milked and isolated in the procedure described above. All isolated colostrum-derived PMNs were stained with 250 ml of Turk's solution (Merck, Berlin) and counted in a Neubauer haemocyte counting chamber. The results were obtained in cells per milliliter and compared using a t-test.
3
Murine Immune Cell Profiling by Flow Cytometry
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Blood was collected in EDTA tubes (BD Pharmingen, San Diego, CA, USA) and leukocytes counted in a Neubauer hemocytometer using Turk’s solution (Merck KGaA, Darmstadt, Germany). Lymphocytes and granulocytes were distinguished by morphology of nuclei. After lysis of red blood cells, FcγII and FcγIII receptors were blocked with anti-CD16/CD32 antibodies at 5 μg/ml (Mouse BD Fc Block™, BD Pharmingen) then specific primary antibodies were added: fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD45R/B220 monoclonal antibody; phycoerythrin (PE)-conjugated hamster anti-mouse CD3e monoclonal antibody; PE-conjugated rat anti-mouse CD335 (NKp46) monoclonal antibody; FITC-conjugated rat anti-mouse CD49b (clone DX5) monoclonal antibody; FITC-conjugated rat anti-mouse CD8a monoclonal antibody; PE-conjugated rat anti-mouse CD4 monoclonal antibody; PE-conjugated rat anti-mouse CD44 monoclonal antibody. All antibodies were from BD Pharmingen. The Partec CyFlow® space cytofluorimeter was used and analysis was performed with Windows™ FloMax® software. Through backgating from the CD3+ and B220+ clusters, we selected the lymphocyte gate in the FSC-SSC dot plot. At least 10,000 events were acquired in the gate. The analysis template was kept constant in the various experiments. The same reagents and procedures were used for splenocyte immunophenotyping.
4
Curcumin-TPGS Diclofenac Sodium Gel
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Curcumin was purchased from PT. Phytochemindo, Reksa, Bogor, Indonesia, d -α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) was commercially obtained from Eastman Chemical Company, Liverpool, UK, diclofenac sodium was purchased from PT Kimia Farma, Bandung, Indonesia, λ-carrageenan was commercially obtained from Sigma-Aldrich, Singapore, NaCl 0.9% was purchased from Otsuka, Tokyo, Japan, Turk’s solution was from Merck, Darmstadt, Germany, heparin was obtained commercially from Inviclot® (Tangerang, Indonesia). All materials used were pharmaceutical grade.
5
Polarization of Peritoneal Macrophages in Mice
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Sterile 3 % thioglycolate (TG) was i.p. injected in C57BL/6 mice. After 5 days, mice were sacrificed, and peritoneal cells (Pec) were recovered by lavage with saline (S.A.L.F, Bergamo, Italy). Pec were centrifuged at 1200 rpm for 10 min at 4 °C. Supernatants were eliminated and pellets re-suspended in RPMI 1640 medium supplemented with penicillin (100 U/ml), streptomicin (100 U/ml), and ultra-glutamine (100 U/ml) (Lonza, Milano, Italy). Cells were counted with Turk’s solution (Merck Chemicals), plated at 5 × 106 in 60-mm petri dishes (Becton Dickinson) and then incubated at 37 °C and 5 % CO2 for 1 h. Finally, cells were washed twice with saline and RPMI 1640 medium supplemented with 10 % FBS, penicillin (100 U/ml), streptomicin (100 U/ml), and ultra-glutamine (100 U/ml) was added. One hour after incubation at 37 °C and 5 % CO2, Pec polarization into M1 or M2 was performed by stimulation with rIFNγ (20 ng/ml, Peprotech, USA) plus LPS (100 ng/ml) for 4 h or rIL4 (20 ng/ml, R&D System, Minneapolis, MN, USA) for 18 h.
6
Differential Blood Cell Counting
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Blood cells obtained by submandibular puncture in heparinized tubes were differentially counted microscopically (mononuclear and polymorphonuclear (PMN) cells) in a Neubauer chamber with Turk's solution (Merck, Kenilworth, NJ).
7
Immunophenotyping of Mouse Blood Cells
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The heparinized blood from each animal was divided into two parts. A volume of 150 µl was designated for blood count and measured using an ABX Pentra 60 C+ hemoanalyzer (Horiba, Kyoto, Japan). The rest of the blood (200–300 µl) was lysed using EasyLyse™ (Dako, Glostrup, Denmark). The amount of cells in suspension was as determined by Turk’s solution (2% acetic acid; Sigma-Aldrich) using a hemocytometer chamber. Cells (5 × 105/100 µl) were marked with two panels of monoclonal antibodies. The first panel was delineated to detect T, B, and NK lymphocytes (CD3ϵ, CD4, CD8, CD19, and NK1.1). The second panel was designed for determination of monocytes and neutrophils (CD11b, F4/80, Ly6C, and Ly6G). The following monoclonal antibodies with fluorochromes for flow cytometry analysis were purchased from BioLegend (San Diego, CA, United States): anti-mouse CD3ϵ–FITC, CD4–BV421, CD19–PE, CD11c–BV421, CD45–APC/Fire™750, F4/80–PE, and from BD Bioscience: CD8–PECy7, CD11b–BV510, Ly6C–FITC, Ly6G–PECy7, and NK1.1–APC.
8
Isolation of Human Neutrophils from Whole Blood
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Whole blood was diluted 1:2 in sterile Dulbecco′s Phosphate Buffered Saline (DPBS) without Ca2+ and Mg2+ and slowly pipetted on top of Pancoll (1.077 g/ml) (PAN-Biotech, Aidenbach, Germany) in a 2:3 volume ratio. The blood was spun down for 30 minutes at 400g without braking. Following centrifugation, the three top layers (i.e. diluted plasma, PBMCs and Pancoll) were carefully discarded, leaving a red pellet with erythrocytes and granulocytes. The pellet was then mixed with equal volumes of DPBS and 6% (w/v) dextran in DPBS, followed by incubation for 30 minutes at 37°C. Next, the supernatant containing neutrophils was transferred to a clean tube, and the cells were washed twice with DPBS (centrifugations were performed for 10 minutes at 177g). Subsequently, the cell pellet was suspended in 5 ml DPBS, and 25 ml sterile ultrapure water was added. After 30 seconds, 10 ml 3.6% (w/v) NaCl was added and mixed by inversion. The cells were spun down and washed with DPBS, whereupon a small aliquot was diluted in Turk’s solution (Sigma-Aldrich) and used to determine the cell concentration using a Bürker chamber. The average yield (± SD) was 1.72 × 106 (± 0.66 × 106) neutrophils/ml blood. The average purity (± SD) of the neutrophils (defined as live CD16+CD66b+ cells) was 95.2 (± 1.6) %.
9
Leukocyte Differential Counting Protocol
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The Neubauer hemocytometer chamber was used for total blood leukocyte count and the blood was diluted (1:10) in Turk's solution (SIGMA, St. Louis, MO, USA). Values are expressed as ×104/mL leukocytes.
Differential counting of circulating leukocytes was performed on slides stained with the Fast Panoptic kit (AppliChem GmbH, Darmstadt, HE, Germany). One hundred leukocytes were counted for each sample by covering fields in a continuous zigzag manner. The smears were examined under a microscope with a 40× objective, identifying leukocytes in neutrophils, eosinophils, basophils, lymphocytes, and monocytes.
Differential counting of circulating leukocytes was performed on slides stained with the Fast Panoptic kit (AppliChem GmbH, Darmstadt, HE, Germany). One hundred leukocytes were counted for each sample by covering fields in a continuous zigzag manner. The smears were examined under a microscope with a 40× objective, identifying leukocytes in neutrophils, eosinophils, basophils, lymphocytes, and monocytes.
10
Fluorescent Staining Reagents for Cellular Analysis
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Fluorophores calcein AM (5–10 μM), Calcein Red-Orange AM (5 μM), LTR (100nM), Fluo-4 (10 μM), Alexa-488–phalloidin, and Rhodamine-phalloidin were purchased from Invitrogen. Human recombinant TNF-α (10–100 ng/mL, BD Biosciences), and BAPTA-AM (100 μM, Invitrogen) were used, as well as FK506 (100 μM), LPS (1–10 mg/kg), cytocholasin D (100 nM), jasplakinolide (100 nM), latrunculin B (50 nM), TB (0.01%), DTT (1 mM), Turk’s solution, and TB, which were purchased from Sigma-Aldrich. Protein A/G-agarose beads were purchased from Santa Cruz Biotechnolgoy Inc. Saponin was purchased from Calbiochem. EZ-Link N-hydroxysuccinimide-SS-biotin (catalog 21331) and streptavidin-Sepharose beads (catalog 20357) were purchased from Thermo Fisher Scientific.
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