Blood was centrifuged in vacutainers used for collection at 2000 rpm for 10 min. Plasma was aspirated from the top and frozen at −80 °C in 1-mL aliquots in cryovials (Thermo Fisher Scientific, cat. #375418) using a freeze controller (Bel-Art Products, cat. #F18844-0000) pre-chilled to −4 °C according to the manufacturer’s instructions. The remaining blood was diluted 1:1 in PBS without calcium or magnesium, layered over 15 mL of Ficoll-Paque (GE Healthcare, cat. #17-1440-03) in an Accuspin tube (Sigma-Aldrich, cat. #A2055), and centrifuged at 2000 rpm for 20 min at 21 °C with acceleration at five and break at zero. The buffy coat leukocyte layer was collected and washed twice in 50 mL of PBS without calcium or magnesium by centrifuging at 2000 rpm for 10 min. Cells were counted, washed again, and resuspended in the Recovery Cell Culture Freezing Medium (Thermo Fisher Scientific, cat. #12648010) at 3.5–10 × 106 cells/mL in 1-mL aliquots, transferred to a freeze controller (Bel-Art Products, cat. #F18844-0000) pre-chilled to −4 °C according to the manufacturer’s instructions, stored at −80 °C for 1–7 days, and then transferred to liquid nitrogen for storage.
Accuspin tube
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
Accuspin tubes are a type of laboratory equipment designed for the separation and collection of samples during centrifugation. They provide a contained and controlled environment for the centrifugation process.
Automatically generated - may contain errors
Lab products found in correlation
Histopaque 1077, by Merck Group (6 mentions)
Tc20 automated cell counter, by Bio-Rad (5 mentions)
Histopaque, by Merck Group (4 mentions)
Ficoll paque, by GE Healthcare (3 mentions)
Cryovial, by Thermo Fisher Scientific (2 mentions)
Recovery cell culture freezing medium, by Thermo Fisher Scientific (2 mentions)
Heparinized tube, by BD (2 mentions)
Vacutainer blood tubes, by BD (2 mentions)
Vacutainer clot activating tubes, by BD (2 mentions)
Histopaque cushion, by Merck Group (2 mentions)
Ficoll paque gradient, by Cytiva (2 mentions)
Rpmi 1640 media, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Macs ls column, by Miltenyi Biotec (1 mentions)
Biofreeze, by Harvard Bioscience (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)
30 protocols using accuspin tube
1
Isolation and Cryopreservation of Blood Cells
2
Isolation of PBMCs from Whole Blood
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PBMCs were isolated from whole blood for an initial global screening of gene transcripts using microarray technology. A similar isolation procedure was employed as described by Boyum [24 (link)]. Briefly, 15 ml of Histopaque-1077 sucrose gradient (Sigma-Aldrich, MO, USA) was pipetted into the upper chamber of an Accuspin Tube (Sigma-Aldrich). The tube was centrifuged (800 × g) for 30 sec to ensure that the Histopaque was below the frit layer. Freshly drawn whole blood was pipetted into the upper chamber of tube. The tube was then centrifuged 800 × g for 15 minutes. The band of mononuclear cells was transferred to an alternate centrifuge tube and washed with 10 ml of isotonic phosphate buffered saline (PBS) three times. Pelleted cells were then resuspended in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 U penicillin & 100 μg streptomycin/ml (Sigma-Aldrich).
3
Plasma and Leukocyte Extraction from Blood
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Blood samples were collected by the standard of care venipuncture; three vacutainers with sodium heparin [BD, cat. #366480] were filled with blood and kept at room temperature until processing, which occurred within 2 h. Blood was centrifuged in vacutainers used for collection at 2000 r.p.m. for 10 min. Plasma was aspirated from the top and frozen at −80°C in 1-mL aliquots in cryovials [Thermo Fisher Scientific, cat. #375418] using a freeze controller [Bel-Art Products, cat. #F18844-0000] pre-chilled to −4°C according to the manufacturer’s instructions. The remaining blood was diluted 1:1 in PBS without calcium or magnesium, layered over 15 mL of Ficoll-Paque [GE Healthcare, cat. #17-1440-03] in an Accuspin tube [Sigma-Aldrich, cat. #A2055] and centrifuged at 2000 r.p.m. for 20 min at 21°C with acceleration at five and break at zero. The buffy coat leukocyte layer was collected and washed twice in 50 mL of PBS without calcium or magnesium by centrifuging at 2000 r.p.m. for 10 min. Cells were counted, washed again and resuspended in Recovery Cell Culture Freezing Medium [Thermo Fisher Scientific, cat. #12648010] at 4.5–11 × 106 cells/mL in 1-mL aliquots, transferred to a freeze controller [Bel-Art Products, cat. #F18844-0000] pre-chilled to −4°C according to the manufacturer’s instructions, stored at −80°C for 1–2 weeks, and then transferred to liquid nitrogen for storage.
4
Isolation of Human Astrocytes and Lymphocytes
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Normal human primary astrocytes were purchased from iXCells Biotechnologies (Cat#10HU-035, San Diego, USA). Cells arrived as passage 4 and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS and EGF. The cell line was stored and re-cultivated as described above.
Peripheral blood samples were collected from three healthy, non-smoking male volunteers without any history of recent disease or exposure to toxic chemical agents. The samples were collected by venipuncture in heparinized tubes (BD, Heidelberg, Germany).
Blood samples were centrifuged (650 g, 10 min, 4°C) immediately after collection. Subsequently, the plasma was removed, the cell suspensions diluted with RPMI 1640 and the lymphocytes isolated by gradient centrifugation (800 g, 16°C min, 16°C) with Histopaque 1077 in Accuspin tubes (Sigma Aldrich, Steinheim, Germany). The cells were collected and washed twice in RPMI 1640 (332 g, 10 min 16°C).
The Ethical Committee of the Medical University of Vienna specifically approved this study.
Peripheral blood samples were collected from three healthy, non-smoking male volunteers without any history of recent disease or exposure to toxic chemical agents. The samples were collected by venipuncture in heparinized tubes (BD, Heidelberg, Germany).
Blood samples were centrifuged (650 g, 10 min, 4°C) immediately after collection. Subsequently, the plasma was removed, the cell suspensions diluted with RPMI 1640 and the lymphocytes isolated by gradient centrifugation (800 g, 16°C min, 16°C) with Histopaque 1077 in Accuspin tubes (Sigma Aldrich, Steinheim, Germany). The cells were collected and washed twice in RPMI 1640 (332 g, 10 min 16°C).
The Ethical Committee of the Medical University of Vienna specifically approved this study.
5
Isolation and Culture of Bovine CD4+ T Cells
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Peripheral blood mononuclear cells were processed using Accuspin tubes (Sigma #A2055). Peripheral blood was diluted with PBS and added to Accuspin tubes that contained Histopaque 1077 (Sigma #10771) under the frit. Cells were centrifuged at 1173 × g for 10 minutes. The buffy coat layer above the frit was added to a new 50 mL tube and was processed as previously described63 (link). Briefly, cells were washed once with PBS at 422 × g 10 minutes. A lyse and restore step was used to remove residual red blood cells. Cells were washed two more times with PBS, counted and sorted for CD4+ T cells.
Bovine CD4+ T cells were sorted using a protocol similar to that which we have previously published63 (link). Briefly, cells were sorted by positive selection using anti-CD4 (WSU, clone: ILA11A), MACs IgG2a+b beads (Miltenyi Biotec #130-047-201) and LS MACS columns (Miltenyi Biotec #130-042-401) according to manufacturer’s instructions. Purity of CD4+ T cells was >90%. Cells were added to RPMI 1640 (ThermoFisher #22400-089), 5% fetal bovine serum, and an antibiotic-antimycotic and incubated at 37 °C for 24 hours.
Bovine CD4+ T cells were sorted using a protocol similar to that which we have previously published63 (link). Briefly, cells were sorted by positive selection using anti-CD4 (WSU, clone: ILA11A), MACs IgG2a+b beads (Miltenyi Biotec #130-047-201) and LS MACS columns (Miltenyi Biotec #130-042-401) according to manufacturer’s instructions. Purity of CD4+ T cells was >90%. Cells were added to RPMI 1640 (ThermoFisher #22400-089), 5% fetal bovine serum, and an antibiotic-antimycotic and incubated at 37 °C for 24 hours.
6
Isolation and Cryopreservation of Lymphocytes
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Peripheral blood samples were collected from three healthy, non-smoking male volunteers without any history of recent disease or exposure to toxic chemical agents. The samples were collected by venipuncture in heparinized tubes (BD, Heidelberg, Germany). Blood samples were collected for comet and MN assays on separate days from the same individuals.
For comet assays, blood samples from three donors were centrifuged (650g, 10 min, 4 °C) immediately after collection. Subsequently, the plasma was removed, the cell suspensions were diluted with RPMI 1640, and the lymphocytes were isolated by gradient centrifugation (800g, 16 °C min, 16 °C) with Histopaque 1077 in Accuspin tubes (Sigma-Aldrich, Steinheim, Germany). The cells were collected and washed twice in RPMI 1640 (332g, 10 min 16 °C), suspended in freezing medium (Biofreeze, Biochrom AG, Berlin, Germany) and stored at −80 °C.
Blood samples for cytokinesis-block micronucleus (CBMN) assays were collected from the same volunteers. The isolation of lymphocytes was performed as described by Fenech (2007 (link)). The cells were cultivated under standard conditions (37 °C, humidified atmosphere, 5 % CO2) in RPMI 1640 medium supplemented with 10 % FCS.
For comet assays, blood samples from three donors were centrifuged (650g, 10 min, 4 °C) immediately after collection. Subsequently, the plasma was removed, the cell suspensions were diluted with RPMI 1640, and the lymphocytes were isolated by gradient centrifugation (800g, 16 °C min, 16 °C) with Histopaque 1077 in Accuspin tubes (Sigma-Aldrich, Steinheim, Germany). The cells were collected and washed twice in RPMI 1640 (332g, 10 min 16 °C), suspended in freezing medium (Biofreeze, Biochrom AG, Berlin, Germany) and stored at −80 °C.
Blood samples for cytokinesis-block micronucleus (CBMN) assays were collected from the same volunteers. The isolation of lymphocytes was performed as described by Fenech (2007 (link)). The cells were cultivated under standard conditions (37 °C, humidified atmosphere, 5 % CO2) in RPMI 1640 medium supplemented with 10 % FCS.
7
Isolation and Cryopreservation of Peripheral Blood Mononuclear Cells
8
Epstein-Barr Virus Transformation of WBCs
9
Isolation and Cryopreservation of PBMCs
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Blood was collected by femoral venipuncture into EDTA, heparin, and clot-activating vacutainer tubes (BD Biosciences). The EDTA plasma and serum tubes were centrifuged at approximately 1,300g at 4°C for 10 minutes; afterward, the upper layer was collected. For isolation of PBMCs, heparin-treated blood and the spun EDTA pellet were diluted with PBS and carefully layered onto a Histopaque cushion within Accuspin tubes (Sigma-Aldrich). The tubes were centrifuged at approximately 800g room temperature for 15 minutes, and the resulting buffy coat was collected. Cells were washed once in R10 (RPMI medium [Gibco] supplemented with 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin solution, and 1% l -glutamine) and treated briefly with ACK lysing buffer (Gibco) to remove any contaminating erythrocytes. PBMCs were then centrifuged at approximately 250g for 10 minutes to eliminate residual thrombocytes, washed twice with R10 medium, and enumerated with a TC20 Automated Cell Counter (Bio-Rad). Cells were cryopreserved in 10% DMSO in FBS. Before flow cytometry, cryopreserved PBMCs were thawed rapidly in a 37°C water bath (BD Biosciences).
10
Isolation of Postvaccination Plasmablasts
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Blood samples were obtained with informed consent from a donor who was vaccinated with Prevnar13®. Peripheral blood mononuclear cells (PBMC) were purified from blood collected in EDTA tubes by density gradient centrifugation in histopaque over Accuspin™ tubes (Sigma Aldrich) according to the manufacturer’s instructions. Blood was collected nine days after vaccination for the plasmablast isolation and used fresh.
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