| Repository ID: | FR-FCM-ZZ9V | Experiment name: | Standardized flow cytometry assay for identificacion of human monocytic heterogeneity and LRP1 expression in monocyte subpopulations | MIFlowCyt score: | 81.75% |
| Primary researcher: | Gustavo Chiabrando | PI/manager: | Gustavo Chiabrando | Uploaded by: | Gustavo Chiabrando |
| Experiment dates: | 2012-12-03 - 2013-07-31 | Dataset uploaded: | Fri Feb 07 12:02:01 -0500 2014 | Last updated: | Mon Mar 17 12:17:40 -0400 2014 |
| Keywords: | [monocyte] [CD14] [Blood] [leukocyte] [lymphocyte] [granulocyte] [monocyte subpopulations] [CD16] [LRP1] [atherosclerosis.] | Manuscripts: | |||
| Organizations: |
Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Bioquímica Clínica, Cordoba, Cordoba (Argentina)
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| Purpose: | To present a novel flow cytometry assay for determination of human blood monocyte subpopulations, i.e., the CD14++CD16-, CD14++CD16+, and CD14+CD16++ monocytes, and LRP1 expression in each monocyte subpopulation. | ||||
| Conclusion: | -LRP1 is mainly expressed in monocytes, which allows a clear separation of these cells by flow cytometry from other leukocyte subsets using a SSC vs. LRP1 plot. -Percentages of monocyte subpopulations measured by the LRP1 monocytic strategy were significantly comparable with those obtained with the HLA-DR procedure. -LRP1 is differentially detected by flow cytometry in monocyte subpopulations, being higher in CD14++-monocytes (classical and intermediate) than in CD14+-monocytes (nonclassical) of healthy subjects. -The total variation yield by the measurement of LRP1 and monocyte subpopulations [(aCV%2 + bCV%2)½] was not higher than 20%. | ||||
| Comments: | None | ||||
| Funding: | Not disclosed | ||||
| Quality control: | To minimize pre-analytical variance as much as possible, specimens were collected at approximately the same time of the day (between 08:00 and 09:30 a.m.). Specimens of each individual were analyzed in duplicate. | ||||
| Sample Type | |
|---|---|
| - Peripheral Blood | Specimen.fcs |
| Description | Fresh whole blood was drawn into EDTA-K3 collection tubes (DVS No. 1213752.5) and specimens were prepared for flow cytometry within 30 min. |
| Sample source: | [ other ] Peripheral Blood |
| Sample characteristic: | Human Monocytes |
| Sample treatment: | About 50 µl of whole blood was added to a 5 ml polystyrene round-bottom tube (No. 352008; BD Biosciences). Lysis of erythrocytes was performed using a lysing buffer (No. 555899; BD Pharm Lyse) for 15 minutes. |
| Description | Fresh whole blood was drawn into EDTA-K3 collection tubes (DVS No. 1213752.5) and specimens were prepared for flow cytometry within 30 min. |
| Sample source: | [ other ] Peripheral Blood |
| Sample characteristic: | Human Monocytes |
| Sample treatment: | About 50 µl of whole blood was added to a 5 ml polystyrene round-bottom tube (No. 352008; BD Biosciences) and 1 µl of anti-CD14 FITC antibody (dilution 1:50) was added. The sample was then incubated for 20 min at 4 °C in the dark. Lysis of erythrocytes was performed using a lysing buffer (No. 555899; BD Pharm Lyse) for 15 minutes. |
| Description | Fresh whole blood was drawn into EDTA-K3 collection tubes (DVS No. 1213752.5) and specimens were prepared for flow cytometry within 30 min. |
| Sample source: | [ other ] Peripheral Blood |
| Sample characteristic: | Human Monocytes |
| Sample treatment: | About 50 µl of whole blood was added to a 5 ml polystyrene round-bottom tube (No. 352008; BD Biosciences) and 1 µl of anti-CD16 APC-Cy7 antibody (dilution 1:50) was added. The sample was then incubated for 20 min at 4 °C in the dark. Lysis of erythrocytes was performed using a lysing buffer (No. 555899; BD Pharm Lyse) for 15 minutes. |
| Description | Fresh whole blood was drawn into EDTA-K3 collection tubes (DVS No. 1213752.5) and specimens were prepared for flow cytometry within 30 min. |
| Sample source: | [ other ] Peripheral Blood |
| Sample characteristic: | Human Monocytes |
| Sample treatment: | About 50 µl of whole blood was added to a 5 ml polystyrene round-bottom tube (No. 352008; BD Biosciences) and 1 µl of anti-CD45 PE-Cy5 antibody (dilution 1:50) was added. The sample was then incubated for 20 min at 4 °C in the dark. Lysis of erythrocytes was performed using a lysing buffer (No. 555899; BD Pharm Lyse) for 15 minutes. |
| Description | Fresh whole blood was drawn into EDTA-K3 collection tubes (DVS No. 1213752.5) and specimens were prepared for flow cytometry within 30 min. |
| Sample source: | [ other ] Peripheral Blood |
| Sample characteristic: | Human Monocytes |
| Sample treatment: | About 50 µl of whole blood was added to a 5 ml polystyrene round-bottom tube (No. 352008; BD Biosciences) and 1 µl of anti-LRP1 PE antibody (dilution 1:50) was added. The sample was then incubated for 20 min at 4 °C in the dark. Lysis of erythrocytes was performed using a lysing buffer (No. 555899; BD Pharm Lyse) for 15 minutes. |
| Description | Fresh whole blood was drawn into EDTA-K3 collection tubes (DVS No. 1213752.5) and specimens were prepared for flow cytometry within 30 min. |
| Sample source: | [ other ] Peripheral Blood |
| Sample characteristic: | Human Monocytes |
| Sample treatment: | About 50 µl of whole blood was added to a 5 ml polystyrene round-bottom tube (No. 352008; BD Biosciences) and 1 µl of each antigen specific fluorochrome-labeled antibody (dilution 1:50) was added. The sample was then incubated for 20 min at 4 °C in the dark. Lysis of erythrocytes was performed using a lysing buffer (No. 555899; BD Pharm Lyse) for 15 minutes. |
| Manufacturer | Becton Dickinson (BD Biosciences) | Default instrument settings used. |
|---|---|---|
| Model | BD FACSCanto II | |
| Optical filter installation dates |
| Manufacturer | Becton Dickinson (BD Biosciences) | Default instrument settings used. |
|---|---|---|
| Model | BD FACSCanto II | |
| Optical filter installation dates |
| Manufacturer | Becton Dickinson (BD Biosciences) | Default instrument settings used. |
|---|---|---|
| Model | BD FACSCanto II | |
| Optical filter installation dates |
| Manufacturer | Becton Dickinson (BD Biosciences) | Default instrument settings used. |
|---|---|---|
| Model | BD FACSCanto II | |
| Optical filter installation dates |
| Manufacturer | Becton Dickinson (BD Biosciences) | Default instrument settings used. |
|---|---|---|
| Model | BD FACSCanto II | |
| Optical filter installation dates |
| Manufacturer | Becton Dickinson (BD Biosciences) | Default instrument settings used. |
|---|---|---|
| Model | BD FACSCanto II | |
| Optical filter installation dates |