The solid pellets were poured into cryovials (Sarstedt, Nümbrecht, Germany), and the microtubes were rinsed with 1 mL of methanol (HPLC-grade, Merck, Darmstadt, Germany), which was added to the cryovial with the sample. In addition, ca. 0.9 g of Lysing Matrix D (Thermo Savant, Illkirch, France) was added to the sample. The cells were lysed by reciprocal shaking at a speed of 4.5 m s−1 for 45 s in a Bio 101 FastPrep Instrument (Thermo Savant, Illkirch, France). Subsequently, the vials were centrifuged for 15 min at 16,100× g (5415 R, Eppendorf, Hamburg, Germany), and 500 µL of the supernatants were transferred into a 0.45 µm spin filter (Merck Millipore, Darmstadt, Germany), which was centrifuged for 30 s at 2300× g. The filtrates were transferred into 2 mL HPLC crimp vials (Agilent Technologies, Waldbronn, Germany). The remaining 500 µL of supernatant was placed into the previously used spin filter, which was centrifuged again for 30 s at 2300× g. Filtrates of the same sample were combined and left to dry overnight in the laboratory fume hood. Dry samples were reconstituted in methanol to a defined volume of 500 µL. Then, the vial was crimped with a silicone septum (11 mm Silver Aluminum Crimp Cap, PTFE/silicone septa, Agilent Technologies, Waldbronn, Germany) and vortexed to ensure the complete solution of all sample constituents.
Cryovials
Manufactured by Sarstedt
Sourced in Germany
Cryovials are laboratory containers designed for the storage of biological samples at cryogenic temperatures. They are made of high-quality materials, such as polypropylene, to ensure sample integrity and long-term preservation.
Automatically generated - may contain errors
Lab products found in correlation
Hplc grade, by Merck Group (1 mentions)
Trilogy fluorometer, by Turner Designs (1 mentions)
Dulbecco s minimum essential medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (1 mentions)
Six well plate, by Corning (1 mentions)
Salivette synthetic swabs, by Sarstedt (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Zirconia silica beads, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
24 well plate, by Corning (1 mentions)
11 protocols using cryovials
1
Bacterial DNA Extraction and Preparation
2
Measuring Biomass Parameters in Aquatic Samples
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Biomass parameters were sampled in triplicate from t0 and from each replicate bottle at tfin. After thoroughly inverting the bottles, we vacuum-filtered (<−200 mbar) 300 mL for chlorophyll a (chla), 200 mL for particulate organic carbon and nitrogen (POC/PON), and the same volume of sterile water for blanks onto pre-combusted glass-fiber filters (GF/F Whatman, Maidstone, UK). These were put into 2 mL cryovials (Sarstedt, Nümbrecht, Germany) and kept at −80 °C until processing. Filters for chla were manually shredded in 6 mL of 90% acetone and extracted for 20 h at 8 °C according to the EPA method 445.0 [45 ]. The extract was centrifuged to remove residual filter snips, and chla was determined on a Trilogy fluorometer (Turner Designs, San Jose, CA, USA) after correcting for phaeopigments via acidification (1 M HCl). Filters for POC/PON were also acidified (0.5 M HCl) and dried for 12 h at 60 °C. Analysis was performed using a gas chromatograph CHNS-O elemental analyzer (EURO EA 3000, HEKAtech, Wegberg, Germany). The chla:POC ratio was calculated by dividing the chla concentration by the POC concentration, and the C:N ratio was calculated by dividing the molar mass of POC by PON.
3
Saliva Preprocessing for Bioanalysis
4
Serum Viral Isolation from Livestock
5
Efficient Cryopreservation of PBMC
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To evaluate T cell responses efficiently we preserved PBMC in liquid nitrogen over a maximum of 6 months. Blood was sampled in CPT tubes for ELISPOT and in EDTA tubes for flow cytometry. PBMC were isolated within 2 h after venipuncture by 1500 g centrifugation for 20 min. After washing, we diluted the PBMC at a concentration of 2x107cells/ml in freezing medium A (60% FCS; 40% RPMI, Biochrom, Berlin, Germany) at 4 °C. The same volume of freezing medium B (20% DMSO, 80% FCS) at 4 °C was added before cell suspensions were transferred into cryovials (Sarstedt, Nürnbrecht, Germany) and set in one at 4 °C prechilled Nalgene Cryogenic Freezing Container (Fisher Scientific, Hannover, Germany) which was placed in −80 °C overnight. After 12-24 h, cryovials were transferred into liquid nitrogen tanks for storage until ELISPOT.
Thawed cell suspensions were transferred into a 15 ml tube containing 10 ml of ice cold PBS. After two washing steps, cells were pipetted in complete RPMI medium (93% RPMI-1640. 5% heat-inactivated FCS, 1% L-glutamin, 1% penicillin-streptomycin) and counted manually using Trypan blue-staining and light microscopy.
Thawed cell suspensions were transferred into a 15 ml tube containing 10 ml of ice cold PBS. After two washing steps, cells were pipetted in complete RPMI medium (93% RPMI-1640. 5% heat-inactivated FCS, 1% L-glutamin, 1% penicillin-streptomycin) and counted manually using Trypan blue-staining and light microscopy.
6
Faecal Sample Processing for Microbiome Analysis
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Fresh faecal samples were collected from 7 individuals (5 females). Participants were healthy adults, free from gastrointestinal conditions, with no antibiotic exposure in the 28 days prior to sample collection. Participants provided written informed consent. Ethical approval was received from the Clinical Research Ethics Committee of the Cork Teaching Hospitals, Cork, Ireland. Fresh samples were collected and aliquoted within 4 hours of defecation. Each sample was homogenised and 250mg aliquots from each faecal sample was added to 3 separate cryovials (Sarstedt, Wexford, Ireland) containing zirconia/silica bead mix (Stratech Scientific, UK). One aliquot per individual was immersed in dry ice for 4 minutes until completely frozen. These ‘snap’ frozen samples were then stored at -80°C for 7 days before DNA extractions and culturing were completed. The ‘frozen’ samples were immediately frozen at -80°C for 7 days following collection and aliquoting. The ‘fresh’ samples were processed within 4 hours of collection, and were stored at 4°C during this brief period between collection and DNA extraction. In the case of culture-based analysis, 1g was taken from each homogenised faecal sample and stored under the 3 storage conditions detailed above, before being used for culturing.
7
Urine collection and storage protocol
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Urine samples were collected at baseline during the visit to the memory clinic in polystyrene containers (Avantor) and centrifuged for 10 minutes at 1800g at 4°C and stored in cryovials (Sarstedt, 1 ml) at −80°C in the Amsterdam UMC Biobank. The effect of freezing/thawing cycli for Nfl in urine is unknown, therefore we used fresh material that had not undergone freeze/thaw cycles before our analysis. Off note: urine was not collected in the morning.
8
Sperm Cryopreservation Optimization
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Both gonads from the same individual were placed in 500 μl FBS solution and shaken for sperm release. A volume equal to 60 μl of the resulting mix was mixed with 10% DMSO or 10% methanol in Eppendorf tubes (or Cryovials, Sarstedt #72.692.005) and incubated for 1 h at 4 °C. Vials were distributed to (1) Mr. Frosty Freezing Container (estimated − 1 °C per min), (2) a glass beaker surrounded by dry ice (estimated − 20 °C per min), (3) direct contact with dry ice (estimated − 50 °C per min), (4) a Dewar vessel partially filled with liquid nitrogen, where the Eppendorf tubes were placed in a box exposed to nitrogen gas phase (estimated − 100 °C per min); and 5) direct liquid nitrogen contact (estimated − 200 °C per min) (Fig. 4 C). Cooling rates were estimated using a digital thermometer with a detection range − 50 °C to + 110 °C with the probe inserted in the tube. Cooling rate was calculated by measuring the temperature difference between room temperature and − 50 °C, divided by the time required to reach − 50 °C. Fifteen minutes after the temperature of − 50 °C or below was reached, vials were removed from their freezing setups and placed in liquid nitrogen overnight. After one day, frozen sperm samples were revived using BSMIS 0.25 × and monitored for activation.
9
Adolescent Saliva Collection Protocol
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In this study, saliva was obtained from adolescents (6-16 years) through a standardized protocol, whereby samples were collected via Salivette synthetic swabs and Cryovials (Sarstedt, Germany) at Ghent University Hospital in the presence of trained staff members. Saliva was collected between 4.30 and 5.30 p.m., with the participants being restrained from any food or drinks (except water) for at least 3 h prior to collection and allowed to brush their teeth in the morning only. Smoking or alcohol use was not permitted during the whole day. Samples were stored at -80 °C. The study was approved by the Ethical Committee of Ghent University Hospital EC UZG 2017/0527.
10
Infectious Virus Titration in Vero Cells
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For analysis of infectious virus from tissues via plaque assay in Vero cells, ½ brain, spleen, both kidneys, heart, liver, and lung were weighed and homogenized in 500 ul of serum-free DMEM or 1x PBS in 2 ml cryovials (Sarstedt) containing 2.3 mm Zirconia/Silica beads (Fisher) on a bead beater at 5300 rpm for 25 seconds. Samples were then clarified at 5000 x g for 10 minutes, and the clarified supernatant transferred to a new tube. Vero cells were plated the previous day in 24-well plates (Corning) at a density of ~1.3 x 105 cells per well. The clarified tissue homogenate supernatants and plasma samples were serially diluted 10-fold in DMEM supplemented with 2% FBS and penicillin-streptomycin. Media was removed from plates, and 200ul of serially diluted sample was plated per well and plates were incubated at 37°C for one hour. After incubation, 0.5 ml of MEM (Gibco) with 1.5% carboxymethyl cellulose (CMC) was overlayed per well, and plates were returned to the 37°C incubator. After 3 days for INKV and SSHV, and 5 days for LACV, TAHV, and JCV, plates were fixed with 10% formaldehyde (Sigma), stained with 0.35% crystal violet, rinsed, air-dried, and plaques counted for each sample and dilution. Sample titers were calculated as PFU/mg tissue or PFU/ml plasma.
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