To determine cellular anti-CD49d saturation levels, venous blood was collected in EDTA tubes after cardiac puncture, and mononuclear cells were enriched using a Histopaque-1077 gradient (Sigma-Aldrich). Cells were diluted to a concentration of 106 cells/ml and stained with a mouse anti-Rat IgG2b PE-conjugated secondary antibody (1:20, clone R2B-7C3; eBioscience) to detect cell-bound anti-CD49d for 30 min at 4°C. For determination of anti-CD49d saturation, cells were incubated with a saturating amount of anti-CD49d (10 µg/ml) for 30 min at room temperature and subsequently stained with the antibody as described above. Stained cells were then analyzed on a BD FACSVerse flow cytometer (BD Biosciences), and analysis was performed using FlowJo software (version 10.0). The anti-CD49d saturation level was then calculated as percentage of the mean fluorescence intensity (MFI) from in vivo bound anti-CD49d from the MFI of in vitro saturated cells by the following equation: The number of anti-CD49d molecules per cell was determined using a PE fluorescence quantitation kit (BD Quantibrite PE, #340495; BD Biosciences) following the manufacturer’s instructions.
Histopaque 1077 gradient
Manufactured by Merck Group
Sourced in United States
Histopaque-1077 is a sterile, endotoxin-tested solution designed for the isolation of mononuclear cells from human peripheral blood by density gradient centrifugation. It is a ready-to-use solution that does not require dilution or adjustment prior to use.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Cd34 microbead kit, by Miltenyi Biotec (3 mentions)
Ls column, by Miltenyi Biotec (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Trypan blue, by Lonza (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Coolcell xl, by Corning (2 mentions)
Cryopro, by Avantor (2 mentions)
Facscanto, by BD (2 mentions)
Collagenase 2 solution, by Thermo Fisher Scientific (2 mentions)
Collagenase type 1, by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Cd105 ad2 clone, by BioLegend (2 mentions)
Cd73 43a3 clone, by BioLegend (2 mentions)
Egm 2 medium, by Lonza (2 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Quantibrite pe, by BD (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
33 protocols using histopaque 1077 gradient
1
Quantifying Anti-CD49d Saturation Levels
2
Isolation and Characterization of PBMCs
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The subjects of this study included healthy blood donors from the University Hospital of the Botucatu Medical School (UNESP), Brazil, with ages ranging from 25 to 50 years. The investigation was carried out according to the Helsinki’s Declaration and approved by the institutional Research Ethics Committee. All the cell donors were informed about the procedures, objectives and risks involved in the study, and signed an agreement form (OF. 17/2007-CEP).
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood of seven healthy adult donors by centrifugation on Histopaque®-1077 gradient (Sigma-Aldrich Inc., USA). Cell suspension containing PBMC was washed twice with RPMI 1640 tissue culture medium (Sigma-Aldrich) and suspended in a complete culture medium [RPMI 1640 supplemented with 2 mM L-glutamine (Sigma-Aldrich), 40 μg.mL−1 gentamicin (Gibco Laboratories, USA) and 10% heat-inactivated autologous human serum]. Monocytes were identified by neutral red staining, and cell suspension was adjusted to 106 monocytes/mL for the assays.
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood of seven healthy adult donors by centrifugation on Histopaque®-1077 gradient (Sigma-Aldrich Inc., USA). Cell suspension containing PBMC was washed twice with RPMI 1640 tissue culture medium (Sigma-Aldrich) and suspended in a complete culture medium [RPMI 1640 supplemented with 2 mM L-glutamine (Sigma-Aldrich), 40 μg.mL−1 gentamicin (Gibco Laboratories, USA) and 10% heat-inactivated autologous human serum]. Monocytes were identified by neutral red staining, and cell suspension was adjusted to 106 monocytes/mL for the assays.
3
Blood Fractionation and Cell Isolation
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Blood culture samples were fractionated using SSTs (Vacuette Z serum Sep. Clot activator, Greiner bio-one, Kremsmunster, Austria #455071) to obtain the plasma fraction. The sample was loaded into the SSTs and centrifuged for 10 min at 1700 g at 20°C. For F. tularensis, the upper fraction was collected, and the bacteria lying on the gel matrix were recovered and added to the plasma fraction. For Y. pestis, the upper fraction was discarded, and the bacteria lying on the gel matrix were recovered with 1 ml of PBS. Mononuclear cells were purified from blood culture samples using Vacutainer CPTs (cell preparation tubes) NC (BD, Sparks, MD, USA, #362781) and Histopaque®-1077 gradient (Sigma, Israel #10771) following the manufacturer’s protocols.
4
Isolation and Culture of Human PBMCs
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Human cells were prepared according to an approved protocol (East London and the City Research Ethics Committee and Queen Mary University of London [QMUL] Animal Welfare & Ethical Review Board; 06/Q605/40; P/00/029). Peripheral blood was collected from 6 different healthy volunteers by intravenous withdrawal in 3.2% sodium citrate solution (1:10). PBMCs were isolated by density centrifugation on a Histopaque-1077 gradient (Sigma-Aldrich) according to the manufacturer’s instructions, and were plated in RPMI 1640 for 1 hour. Cells were washed 3 times with ice-cold PBS without Ca2+/Mg2+ to remove lymphocytes, and adherent cells were incubated in RPMI 1640 containing 20% heat-inactivated FCS for 14 days.
5
Isolation and Stimulation of PBMCs for Malaria Research
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Peripheral blood mononuclear cells (PBMCs) from the blood of malaria-naïve healthy volunteers were isolated by density centrifugation through a Histopaque-1077 gradient (Sigma-Aldrich) and suspended in RPMI-1640 medium supplemented with 10% FCS (Gibco), 1mM sodium pyruvate, 1x penicillin/streptomycin, 2mM L-glutamine and 2-mercaptoethanol (Invitrogen). PBMCs were plated in a 24-well plate (Nunc) at 5x105 per well and incubated overnight in a 5% CO2 incubator set at 37°C. The following day, the isolated PBMCs were exposed to P. falciparum (W2)-enriched schizonts for 48 hours at different ratios. Naïve RBCs were added as a negative control at a ratio of 40:1. In some experiments, isolated PBMCs were depleted of T cells using human CD3 microbeads following the manufacturer’s instructions (Miltenyi) after being exposed to P. falciparum (3D7) schizont-stage parasites. Stimulated PBMCs were stored in RNA Stat60 (Tel-Test Inc) at -20°C until use.
6
Transwell Migration Assay for Bone Marrow Cells
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Whole population of bone marrow cells was obtained from wt and Pax7−/− mice. Bone marrow cells were washed in phosphate buffered saline (PBS) and centrifuged in Histopaque 1077 gradient (Sigma‐Aldrich) to remove erythrocytes. Obtained mononucleated cells were seeded on culture inserts (8 µm pore size) coated with Matrigel containing reduced growth factor concentration (Becton Dickinson Bioscience) and cultured in α‐MEM (Sigma‐Aldrich) supplemented with 2 mM l ‐glutamine (Life Technologies), 20% FBS, and 1% antibiotic (penicillin and streptomycin, Life Technologies). After 48 h of culture, non‐adherent cells were removed. Next, culture medium present in the lower chamber was supplemented with Sdf‐1 (50 ng/mL). After an additional 48 h of culture, the cells were fixed with methanol and stained with Giemsa (Merck, Darmstadt, Germany), according to the manufacturer's protocol. The number of cells that migrated through the insert pores and localized either at the insert surface facing the lower dish or at the bottom of the lower chamber was counted. Analysis was performed in triplicates.
7
Isolation and Differentiation of BMMCs and BMDMs
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BMMCs were separated from whole BMC preparations flushed from WT or CD11b/LIF femurs and tibiae and separated using a histopaque-1077 gradient (Sigma). The freshly-isolated BMMCs were then used for RNA isolation and analysis. For preparation of BMDMs, BMCs were aseptically flushed from WT or CD11b/LIF femurs and tibiae and differentiated in vitro to BMDMs24 (link). BMDMs were stimulated for 24 h with activation media consisting of Dulbecco’s Modified Eagle Medium (DMEM) with 0.25% heat-inactivated fetal bovine serum (FBS; Omega), 100 U/ml penicillin, 100 µg/ml streptomycin (1% P/S), and 10 ng/ml macrophage colony stimulating factor (MCSF; R&D).
8
Isolation and Cryopreservation of CD34+ Cells
9
Isolation of Muscle-Derived Leukocytes
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Leukocyte isolation from the hindlimb (pool of gastrocnemius, extensor digitorum longus, quadriceps and tibialis anterior muscles) and triceps muscles was performed as previously described [44 (link)]. Briefly, the muscles were dissected and kept in cold DMEM (Dulbecco’s Modified Eagle’s Medium) (Corning, 10-013-CV) with 10% FBS (fetal bovine serum) (Hyclone/Cytiva SH30396.03). The muscles were then minced, transferred to 10 ml of digestion media (DMEM with 0.2 mg/ml collagenase P (Roche, Sigma, 11213873001) and 0.02 mg/ml DNAse I (Roche, Sigma, 10104159001)) and incubated for one hour at 37°C. The resulting cell suspension was strained in 100, 70 and 40 μm strainers and separated in the Histopaque-1077 gradient (Sigma, H8889) to isolate the immune cells. The cell suspension was resuspended in 1X PBS/2% FBS.
10
Isolation of CXCR4+ Bone Marrow Cells
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A whole bone marrow cell population was obtained from the tibialis and femoris bone of 3-month-old male C57BI6N-lacZ mice expressing β-galactosidase (β-gal). Bone marrow cells were washed from bones using phosphate-buffered saline (PBS) and centrifuged in Histopaque 1077 gradient (Sigma-Aldrich) to remove erythrocytes. BMCs expressing CXCR4 (CXCR4+BMCs(β-gal+)) were then selected using magnetic columns with antibody against CXCR4 and rabbit secondary antibody conjugated with paramagnetic particles accordingly to the manufacturer’s instructions (Miltenyi Biotec).
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