PB samples were collected through venipuncture in 2 mL BD Vacutainer® spray-coated K2EDTA blood collection tubes (BD Biosciences, San Jose, CA, USA; #367841). One hundred μL of whole blood was transported into 5 mL Corning™ Falcon™ Round-Bottom Polystyrene Tubes (#352054) and stained with 12 monoclonal antibodies against the surface markers: CD45-APC-H7 (clone 2D1, #560178); CD3-PerCP (clone SK7, #340663); CD4-BV510 (clone SK3; #562970); CD8-PE (clone HIT8a, #555635); CD14-BV605 (clone M5E2, #564054); CD16-APC (clone B73.1, #561304); CD56-APC-R700 (clone NCAM16.2, #565139); CD25-PE-CF594 (clone M-A25, #562403); CD11b-BV786 (clone D12, #742642); CD183 (CXCR3)-BV421 (clone 1C6/CXCR3, #562558); CD194 (CCR4)-BV650 (clone 1G1, #744140); and CD196 (CCR6)-BB515 (clone 11A9, #564479) (all from BD Biosciences). The staining procedure was performed in BD Horizon™ Brilliant Stain Buffer (BD Biosciences, #563794) followed by red blood cells lysis with 1x BD FACS™ lysing solution (BD Biosciences, #349202) as suggested by the manufacturer for the Lyse-no-Wash protocol.
Falcon round bottom polystyrene tubes
Manufactured by Corning
Sourced in United States
The Falcon™ Round-Bottom Polystyrene Tubes are a versatile laboratory product designed for a variety of applications. These tubes feature a round bottom shape and are made of polystyrene material. The tubes are available in various sizes to accommodate different volume requirements.
Automatically generated - may contain errors
Lab products found in correlation
Horizon brilliant stain buffer, by BD (1 mentions)
Cd45 apc h7, by BD (1 mentions)
Cd4 bv510, by BD (1 mentions)
Cd3 percp clone sk7, by BD (1 mentions)
Cd8 pe clone hit8a, by BD (1 mentions)
Cd14 bv605, by BD (1 mentions)
Cd25 pe cf594, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Accuri c6, by BD (1 mentions)
Cd4 apc cy7, by BD (1 mentions)
Cd33 apc, by BD (1 mentions)
Cd45 pe, by R&D Systems (1 mentions)
Cytofix fixation buffer, by BD (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Cd3 pe cy7, by BD (1 mentions)
Cell strainer snap cap, by Corning (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
5 protocols using falcon round bottom polystyrene tubes
1
Comprehensive Immunophenotyping of Peripheral Blood
2
PBMC Isolation and Compound Toxicity
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Peripheral blood mononuclear cells (PBMC) were isolated immediately from fresh blood as previously described [43 (link)]. After isolation, PBMC were used to conduct experiments which assessed toxicity of compounds and their impact on proliferation and cytokine production.
The cell suspensions (1 × 106 cells/mL) in culture medium (5% fetal bovine serum—FBS—Euroclone SpA, Pero, Ml, Italy, in RPMI 1640—Biomed Lublin, Lublin, Poland) were added to 5 mL tubes (Falcon® Round Bottom Polystyrene Tubes, Corning, NY, USA) in all experiments. Compounds9 –22 and ibuprofen (IBU) were initially dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and afterwards, were added in appropriate amounts to the cell culture. Final concentrations of compounds and IBU in the cultures were 10, 50 and 100 μg/mL in all biological assays. The maximum concentration of DMSO in the individual assay was lower than 0.5%. These concentrations allowed us to avoid cell culture toxicity. The ibuprofen was selected as the reafference medicine generally used as nonsteroidal anti-inflammatory drugs (positive control). Additionally, control samples contained DMSO in the highest used dose (negative control) were prepared.
The cell suspensions (1 × 106 cells/mL) in culture medium (5% fetal bovine serum—FBS—Euroclone SpA, Pero, Ml, Italy, in RPMI 1640—Biomed Lublin, Lublin, Poland) were added to 5 mL tubes (Falcon® Round Bottom Polystyrene Tubes, Corning, NY, USA) in all experiments. Compounds
3
Optimizing Transduction Efficiency in K562 and CD34+ Cells
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Human K562 cells (1 × 105) and primary human CD34+ cells (5 × 104) were seeded in Falcon round bottom polystyrene tubes (Corning, NY, USA). Cells were resuspended in DMEM before transduction. scAAV6 vectors expressing the EGFP reporter gene under the control of a cytomegalovirus (CMV) enhancer-CBA were either mock-treated or pre-incubated with PVA concentration ranging from 0.001% to 3% and used to transduce cells in triplicates under identical conditions. DMEM was replaced by culture medium 2 h post-transduction. EGFP expression was determined 48 h post-transduction using flow cytometry (Accuri C6, Beckton Dickinson, Franklin Lakes, NJ, USA), followed by processing with software FCS Express 6 Flow.
4
Phospho-specific Flow Cytometric Analysis of PBMC Signaling
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Freshly thawed PBMCs were resuspended in serum-free, antibiotic-free RPMI 1640 media. Cells were distributed (0.5 x 106cells in 100 μL per tube) to Falcon polystyrene round-bottom tubes (12 x 75 mm; Corning Incorporated, Durham, NC) and treated with IL-6 or IFN-γ (100 ng/mL) (R&D Systems, Minneapolis, MN) for 15 min at 37°C before subjecting them to phospho-specific flow cytometric analysis as described previously [7 (link)]. Briefly, after stimulation, cells were fixed by incubating in 2% PFA (BD Cytofix Fixation Buffer; BD Biosciences) for 10 min at 37°C and pelleted. They were then permeabilized by resuspending with vigorous vortexing in 300 μL ice-cold methanol. Cells were washed in staining media. Fluorophore-specific MAbs were added and incubated for 30 min at RT. The following markers were analyzed: CD3-PE/Cy7, CD4-APC/Cy7, CD45RO-PerCP/Cy5.5, CD33-APC, STAT1 (pY701)-AF488, STAT3 (pY705)-AF488, (BD Biosciences, San Diego, CA); and CD45-PE (R&D Systems, Minneapolis, MN). The cells were washed with staining media and pelleted. Finally, the samples were resuspended in 250 μL of staining media and analyzed.
5
PBMC Isolation for Flow Cytometry
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5 ml Falcon polystyrene round-bottom tubes (Corning Life Sciences, cat. no. 352054).
5 ml Falcon polystyrene round-bottom tube, with cell strainer snap cap (Corning Life Sciences, cat. no. 352235).
PBS as staining buffer.
Peripheral blood mononuclear cells (PBMCs) from a young healthy male adult was isolated from a leukocyte reduction chamber (LRC) purchased from the Oklahoma Blood Institute, with Lymphoprep (StemCell Technologies). Oklahoma Blood Institute performed relevant informed consents for blood product use for research purposes. Because the chambers are a byproduct of platelet donation and do not cause additional risk to the donor, the ECU Institutional Review Board does not require board review of protocols. Protocols were approved by East Carolina University Institutional Biosafety Committee protocol #01–19. All the methods were carried out in accordance with relevant guidelines and regulations.
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