Whole blood was collected by venipuncture into either heparin vacutainers (BD, Franklin Lakes) for whole blood experiments or acid-citrate-dextrose (ACD) vacutainers (BD, Franklin Lakes) for platelet isolation studies. For platelet isolation, samples were supplemented with apyrase (Sigma, St Louis) and prostaglandin E1 (Cayman Chemical, Ann Arbor) and PRP was prepared by centrifugation of the ACD whole blood for 20 min at 200×g. Platelets were isolated from the PRP by centrifugation for 10 min at 1000×g and re-suspended in modified Tyrode’s buffer twice at desired platelet counts and kept at 37 °C until used. Isolated platelets were used within 2 h of preparation.
Heparin vacutainers
Manufactured by BD
Sourced in United States
Heparin vacutainers are blood collection tubes that contain the anticoagulant heparin. The function of heparin vacutainers is to prevent blood from clotting during the collection and transportation process, allowing for accurate analysis of the collected sample.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin b, by Gemini Bio (4 mentions)
Aim 5 albumax bsa, by Thermo Fisher Scientific (4 mentions)
Ficoll paque plus, by GE Healthcare (4 mentions)
Sst tube, by BD (3 mentions)
Sepmate, by STEMCELL (3 mentions)
Apyrase, by Merck Group (1 mentions)
Prostaglandin e1, by Cayman Chemical (1 mentions)
Acd vacutainer, by BD (1 mentions)
Dynabeads untouched human nk cells kit, by Thermo Fisher Scientific (1 mentions)
Lymphocyte separation medium, by Lonza (1 mentions)
Vascular endothelial growth factor (vegf), by Bio-Techne (1 mentions)
Rna 6000 nano labchip kit, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
15 protocols using heparin vacutainers
1
Whole Blood and Platelet Isolation
2
SARS-CoV-2 Vaccine Immunogenicity Monitoring
3
Isolation of NK Cells from Breast Cancer Patients
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Peripheral blood of patients with breast cancer and healthy donors was directly obtained in heparin vacutainers, BD Biosciences, San Jose, CA, USA. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using lymphocyte separation medium, Lonza Walkersville, Inc. Houston TX, USA. The cell pellet was washed twice in calcium- and magnesium-free Dulbecco’s PBS-2% FCS-2 mM EDTA, enumerated on a haemocytometer with trypan blue and resuspended at 5 × 107/500 μl. NK cells were isolated through immunomagnetic negative selection using Dynabeads® Untouched™ Human NK Cells kit, Invitrogen Cergy-Pontoise, France, following the manufacturer’s recommendations. The purity (% of CD3−CD56+) of NK cells measured by flow cytometry confirmed that these cells were more than 98% CD3−CD56+ NK cells and less than 2% CD3+.
4
Longitudinal Assessment of Immune Responses
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Venous blood was collected by standard phlebotomy. Blood collection occurred at baseline, approximately one week after first vaccination (“Post 1st dose”), prior to the second vaccination (“Pre 2nd dose”), approximately one week after the second vaccination (“Post 2nd dose”), and one month after the second vaccination (“One month post 2nd dose”), as depicted in Fig. 1A . Peripheral blood mononuclear cells (PBMC) were isolated from heparin vacutainers (BD Biosciences) that were stored overnight at room temperature (RT), followed by processing using Sepmates (Stem Cell, Inc) in accordance with the manufacturer’s recommendations. Serum was collected in SST tubes (BD Biosciences) and frozen immediately at −80°C.
5
Blood Sampling and PBMC Isolation
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Venous blood was collected by standard phlebotomy. Blood collection occurred at baseline, approximately one week after first vaccination (“Post 1st dose”), prior to the second vaccination (“Pre 2nd dose”), approximately one week after the second vaccination (“Post 2nd dose”), and one month after the second vaccination (“One month post 2nd dose”), as depicted in Fig. 1A
6
Plasma Isolation and Neutrophil Extraction
7
Reconditioning of Decellularized Vein Grafts
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Three of the five DC pig veins and three DC human veins were reconditioned.
One of the human veins was split and reconditioned with blood from
two different donors. Before the reconditioning of the ECM, frozen
DC veins were thawed at room temperature or 4 °C overnight. Human
vein femoralis were examined for valve direction to decide on the
flow in blood reactors. All veins were connected to ligands before
the reconditioning started.
Reconditioning of veins with whole
blood has previously been described by Håkansson et al.18 (link) Briefly, 30–50 mL of whole blood from
human or pig donors were collected in heparin vacutainers (BD). The
blood was mixed with STEEN solution (XVIVO Perfusion) 1:1 and 0.5%
AA (Thermo Fisher). The vascular endothelial growth factor (VEGF,
80 ng/mL, Bio-Techne) and fibroblast growth factor 2 (FGF2, 10 ng/mL,
CellGenix) were added to the solution together with 5 μg/mL
acetylsalicylic acid (Sigma-Aldrich) to prevent thrombosis. Glucose
levels were monitored and adjusted to 3–8 mM during the entire
perfusion process (Figure 7 ). After 7 days, veins were washed, and samples were sent
for proteomics analysis. The blood used for reconditioning was species-specific
to the vein segment.
One of the human veins was split and reconditioned with blood from
two different donors. Before the reconditioning of the ECM, frozen
DC veins were thawed at room temperature or 4 °C overnight. Human
vein femoralis were examined for valve direction to decide on the
flow in blood reactors. All veins were connected to ligands before
the reconditioning started.
Reconditioning of veins with whole
blood has previously been described by Håkansson et al.18 (link) Briefly, 30–50 mL of whole blood from
human or pig donors were collected in heparin vacutainers (BD). The
blood was mixed with STEEN solution (XVIVO Perfusion) 1:1 and 0.5%
AA (Thermo Fisher). The vascular endothelial growth factor (VEGF,
80 ng/mL, Bio-Techne) and fibroblast growth factor 2 (FGF2, 10 ng/mL,
CellGenix) were added to the solution together with 5 μg/mL
acetylsalicylic acid (Sigma-Aldrich) to prevent thrombosis. Glucose
levels were monitored and adjusted to 3–8 mM during the entire
perfusion process (
for proteomics analysis. The blood used for reconditioning was species-specific
to the vein segment.
8
Whole Blood IFN-I Response Measurement
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pDC IFN-I was measured via whole blood stimulation as previously published (22 (link)). Briefly, blood was collected immediately following vaccination directly into heparin vacutainers (BD, Franklin Lakes, NJ, USA) containing R10 + Brefeldin A + CL097 and maintained at 37°C in a dry block heater during laboratory transport before further incubation up to 6 hours, red blood cell (RBC) lysis, and staining for flow cytometry via LSR II (flow cytometry antibodies are detailed in Supplementary Table S3
9
Isolation of Human PBMCs from Whole Blood
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Example 4
Whole blood (Stanford Hospital Blood Center, Stanford, Calif.) was collected from individual or mixed donors (depending on the amount of blood required) in 10 mL Heparin vacutainers (Becton Dickinson). Approximately 10 ml of whole anti-coagulated blood was mixed with sterile phosphate buffered saline (PBS) buffer for a total volume of 20 ml in a 50 ml centrifuge tube (PBS, pH 7.4, without Ca+2 and Mg+2). The blood/PBS (20 ml) was layered on top of 15 mL of Ficoll-Paque PLUS (GE Healthcare) in a conical centrifuge tube gently, and the sample was centrifuged at 400×g for 30-40 min at room temperature. The layer of cells containing peripheral blood mononuclear cells (PBMC) at the diluted plasma/Ficoll interface was removed, washed, and centrifuged at 200×g for 10 min at room temperature. Cells were counted with a hemocytomter. The PBMC were washed once with CAR-T media (AIM V-AlbuMAX(BSA) (Life Technologies), with 5% AB serum and 1.25 μg/mL amphotericin B (Gemini Bioproducts, Woodland, Calif.), 100 U/mL penicillin, and 100 μg/mL streptomycin) and used for experiments or were frozen at −80° C.
10
PBL-Derived RNA Isolation and Purification
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The materials and methods used to isolate and purify PBL-derived RNA from all 16 animals have been described by us previously. Briefly, whole blood was collected from each animal in 8 ml heparin vacutainers® (Becton-Dickinson Ltd., Dublin, Ireland) and RNA extraction was performed within 2 h of blood collection. The complete methods used for blood collection, PBL isolation, and total RNA extraction and purification have been described by us previously (12 (link)). RNA quantity and quality checking was performed using the NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip kit (Agilent Technologies, Cork, Ireland). All samples displayed a 260/280 ratio >1.8 and RNA integrity numbers (RIN) >8.0.
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