Synchronization was performed by enriching late stage parasites using Histodenz (Sigma Aldrich) as described in [40 (link)]. Briefly, parasites were resuspended in 5ml complete media and layered on top of 5 ml of 55% Histodenz in complete culture media in a 15 ml tube (Greiner). The mixture was then centrifuged for 3 minutes at room temperature, 1500 g, acceleration 3 and brake 1, resulting in late stage parasites becoming enriched at the interface. For inhibition assays, these parasites were returned to culture, and assays set up after reinvasion had occurred in the subsequent cycle. For protein extracts, immunofluorescence assays and transfections, this was repeated over three consecutive cell cycles to create very tightly synchronized parasites, with schizont samples from a fourth cycle of Histodenz purification used for subsequent analysis.
15 ml tube
Manufactured by Greiner
Sourced in Austria, Germany
The 15 mL tube is a laboratory equipment item used for general storage and sample collection purposes. It has a capacity of 15 milliliters.
Automatically generated - may contain errors
Lab products found in correlation
Neisseria selective medium plus agar plates ns agar, by Thermo Fisher Scientific (2 mentions)
Floqswab, by Copan (2 mentions)
Histodenz, by Merck Group (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
May gr nwald stain, by Merck Group (1 mentions)
Giemsa staining, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Pb 11 p10.1m, by Sartorius (1 mentions)
9 protocols using 15 ml tube
1
Synchronization of Late Stage Malaria Parasites
2
Serum Biomarker Isolation in Hematological Malignancies
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Ten mL of peripheral blood were collected from patients with lymphoma, acute myeloid leukemia (AML), multiple myeloma (MM) and healthy controls after written consent as described before [4 (link)]. Isolation of serum samples and the study were specifically approved by the Ethic Committee of RWTH Aachen University (Permit Number: EK155/09). Blood samples taken before chemotherapy (BC) and during leukopenia after HSCT (AC) were transferred into a 15 mL tube (Greiner, Kremsmünster, Austria), agitated horizontally at 37°C for 1 h to allow coagulation, then incubated upright at 4°C for 4 h and finally centrifuged at 840 x g for 15 min [26 (link)]. Serum supernatant was aliquoted and stored at -80°C until use. Further information on serum samples and patients is provided in Tables A, B, C and D in S1 File .
3
Isolation and Cryopreservation of PBMCs
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PBMCs were isolated from the same blood samples (30 µl mouse blood, 50 µl hamster blood, or 1 ml dog blood) used for pharmacokinetic analysis. After plasma separation as described in the section above (Blood sampling and preparation of plasma samples), the remaining red blood cells were lysed by adding 1 volume of red blood cell (RBC) lysis buffer (Thermo Fisher Scientific, Waltham, MA, United States) (300 µl for mice, 500 µl for hamster, and 10 ml for dogs). The samples were incubated for 15 min at RT and centrifuged at 500 x g for 5 min (RT). The supernatant was removed completely, and the pellet was washed twice with PBS. The PBMC cell pellets were store at −80°C until further analysis. PBMCs of 8 ml human blood were isolated using CPTTM tubes. After a 20-min centrifugation at 1,650 x g at room temperature, the plasma was collected for PK analysis as described in the section above (Blood sampling and preparation of plasma samples). The PBMC layer was collected in a 15 ml tube (Greiner Bio-One, Frickenhausen, Germany), and washed twice with PBS by centrifugation at 300 x g for 10 min at RT. The PBMC pellets were store at −80°C until further PD assessment.
4
Oropharyngeal Sampling and Culturing Protocol
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In the fall of 2018, saliva and oropharyngeal swabs were collected from college students of Hogeschool Utrecht (n = 300). After signing informed consent, students self-collected saliva by spitting 1 ml into a 15 ml tube (Greiner, Kremsmünster, Austria). Next, a study nurse swabbed student’s posterior pharyngeal wall with a nylon swab (FLOQSwabs, COPAN, Brescia, Italy) to collect an oropharyngeal sample. Immediately after collection, saliva (approximately 50 µl) and oropharyngeal swab were used to inoculate Neisseria Selective Medium PLUS agar plates (NS-agar, Oxoid, Badhoevedorp, the Netherlands) and within 20 min plates were placed in a 37 °C, 5% CO2 incubator. Once all samples have been collected, cultured plates were transported at room temperature to the laboratory.
5
Cytospins and Pappenheim Staining
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To perform cytospins, cells were detached from the culture flasks with a trypsin-EDTA solution (0,05% porcine trypsin; 0,02% EDTA (Sigma-Aldrich) and incubated for 5 min at 37°C. 9 mL FACS buffer was added to block the enzymatic reaction and the detached cell suspension was transferred into a fresh 15 mL tube (Greiner) and centrifuged at 300 g for 5 min 500 μL of the obtained cell suspension containing 100.000 cells were added to a cytofunnel of a cytocentrifuge. Cells were centrifuged at 600 rpm for 5 min to spin the cells down on a slide. The supernatant was discarded and the slides were centrifuged at 1100 rpm for 3 min. Slides were air dried prior to staining. To perform Pappenheim staining, slides were stained for 4 min with May-Grünwald stain (Merck), MilliQ water was added and the slides were incubated for 4 min. Slides were washed with MilliQ water and Giemsa staining (Merck) (1:10) was performed for 10 min. Afterwards the slides were washed with MilliQ water and a coverslip was mounted.
6
Bronchoalveolar and Peritoneal Cell Collection Protocols
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Bronchoalveolar lavage was collected as described elsewhere [21 (link)]. A 19-gauge needle hub was inserted into the trachea and 3 mL of ice-cold phosphate-buffered saline (PBS) and 10% fetal bovine serum (FBS) were administered and aspirated slowly through the needle hub. After transfer into a 15 mL tube (Greiner Bio-One, Kremsmünster, Austria), the fluid was kept on ice. The procedure was repeated four times.
For collection of peritoneal cells, we used a protocol adapted from [22 (link)]. Briefly, the peritoneum was exposed and 5 mL of ice-cold PBS with 3% FBS was injected into the peritoneal cavity through a needle. A gentle massage to the abdomen was applied to dislodge any attached cells into the PBS solution. The suspension was collected through a 22-gauge needle into a syringe and transferred into a 15 mL tube, then kept on ice. The above-described procedure was repeated five times.
For collection of peritoneal cells, we used a protocol adapted from [22 (link)]. Briefly, the peritoneum was exposed and 5 mL of ice-cold PBS with 3% FBS was injected into the peritoneal cavity through a needle. A gentle massage to the abdomen was applied to dislodge any attached cells into the PBS solution. The suspension was collected through a 22-gauge needle into a syringe and transferred into a 15 mL tube, then kept on ice. The above-described procedure was repeated five times.
7
Oropharyngeal Sampling of College Students
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In the fall of 2018, saliva and oropharyngeal swabs were collected from college students of Hogeschool Utrecht (n=300). After signing informed consent, students self-collected saliva by spitting 1ml into a 15ml tube (Greiner, Kremsmünster, Austria). Next, a study nurse swabbed student's posterior pharyngeal wall with a nylon swab (FLOQSwabs, COPAN, Brescia, Italy) to collect an oropharyngeal sample. Immediately after collection, saliva (approximately 50 µl) and oropharyngeal swab were used to inoculate Neisseria Selective Medium PLUS agar plates (NS-agar; Oxoid, Badhoevedorp, the Netherlands) and within 20 minutes plates were placed in a 37°C, 5% CO2 incubator. Once all samples have been collected, cultured plates were transported at room temperature to the laboratory.
8
Platelet Lysate Preparation Protocol
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PL was prepared as described previously (Prins et al., 2009 ). Briefly, pooled platelet products containing approximately 1 × 109 thrombocytes/ml were purchased from the Sanquin Blood Bank (Nijmegen, the Netherlands). The product was divided into 5‐ml aliquots in 15‐ml tubes (Greiner Bio‐One), subjected to one freeze/thaw (−80°C/37°C) cycle and stored at −80°C until use. Before adding to the medium, PL was thawed and centrifuged at 2,000 g for 10 min to remove remaining platelet fragments.
9
Monitoring pH-Alterations by BG Particles
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In order to monitor pH-alterations induced by BG particles as a correlate of ion release and bioactivity, the pH was measured from the ODM containing either Vitoss or Vitoss BA scaffolds. 1 ml ODM was transferred to 15 ml tubes (Greiner Bio One, Frickenhausen, Germany) and underwent immediate pH measurement using a benchtop pH-meter (PB-11-P10.1M, Sartorius, Göttingen, Germany).
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