Cryopreserved PBMCs were thawed in a water bath at 37˚C, washed with RPMI-1640 (Sigma-Aldrich; Merck KGaA) supplemented with 2% fetal bovine serum (Sigma-Aldrich; Merck KGaA), and centrifuged at 350 x g for 5 min at room temperature. Cell pellets were resuspended in RPMI-1640, filtered through a 40-µm nylon cell strainer (Corning; Corning, Inc.), and aliquoted at 50 µl into 5 ml polystyrene tubes (up to 3x105 cells per tube). Cells were incubated with fluorochrome-conjugated monoclonal antibodies for 30 min at 4˚C protected from light in an appropriate dilution of 0.5 µg per 106 cells. Following incubation, any unbound antibodies were washed away with 2 ml of cell wash buffer (BD Biosciences) by centrifugation at 350 x g for 5 min at 4˚C. Prior to analysis, cells were gently resuspended in 300 µl of cell wash buffer.
Cell wash buffer
Manufactured by BD
Sourced in United States
Cell wash buffer is a laboratory reagent used to wash and prepare cells for various analytical procedures. It is designed to maintain the integrity and viability of cells during the washing process, ensuring the cells are ready for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Human msc analysis kit, by BD (2 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Facscalibur, by BD (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Optilyse c, by Beckman Coulter (1 mentions)
Navios ex flow cytometer, by Beckman Coulter (1 mentions)
Cd34 pe, by BD (1 mentions)
Pharm lyse lysing buffer, by BD (1 mentions)
Cd274 fitc, by BD (1 mentions)
Cd45 percp, by BD (1 mentions)
Cd4 percp, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Cd25 apc, by BD (1 mentions)
Cd127 fitc, by BD (1 mentions)
Hla dr percp, by BioLegend (1 mentions)
Calibrite beads, by BD (1 mentions)
13 protocols using cell wash buffer
1
PBMC Preparation and Immunostaining
2
Quantification of Neutrophil TLR9 Expression
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To assess TLR9 protein expression in neutrophils, 1 × 106 neutrophils were fixed in 4% paraformaldehyde (PFA), permeabilized in methanol and stained with PE-labelled rat anti-human TLR9 antibody (clone eB72-1665, BD Pharmingen) in 0.1% Triton X-100 and 3% FCS in cell wash buffer (BD) for 30 min in the dark. After washing two times with 0.1% Triton X-100 and 3% FCS in cell wash buffer (BD), cells were analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec) or FACSCalibur (BD). For this, neutrophils were gated using forward and side scatter. For surface TLR9 staining, non-permeabilized cells were incubated with the same antibody in cell wash buffer with 3% FCS for 30 min on ice. Cells were washed twice and immediately analyzed by flow cytometry.
3
Multiparameter Flow Cytometry of Bone Marrow
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Flow cytometry analysis was performed immediately after bone marrow collection. Cells were incubated with appropriate antibodies for 20 min at room temperature. To remove erythrocytes, the cells were incubated with BD Pharm Lyse lysing buffer (BD Biosciences). After being washed in Cell Wash Buffer (BD Biosciences), the cells were suspended in Cell Wash Buffer and analyzed using a BD FACSCanto flow cytometer (BD Biosciences). Gate parameters dividing negative from positive cells were chosen based on isotype antibody control probes.
Tregs were detected based on the CD4 þ CD25 high CD127 - phenotype using the following antibodies: CD4-PerCP, CD25-APC, CD127-FITC (BD Biosciences). We always calculated the percentage of Tregs among all CD4 þ cells. Subpopulations of HSCs (CD45 þ CD34 þ phenotype) exhibiting high expression of CD274 or CD47 markers were identified using CD34-PE, CD45-PerCP, CD47-Alexa Fluor 647, and CD274-FITC antibodies (all antibodies were obtained from BD Biosciences).
Tregs were detected based on the CD4 þ CD25 high CD127 - phenotype using the following antibodies: CD4-PerCP, CD25-APC, CD127-FITC (BD Biosciences). We always calculated the percentage of Tregs among all CD4 þ cells. Subpopulations of HSCs (CD45 þ CD34 þ phenotype) exhibiting high expression of CD274 or CD47 markers were identified using CD34-PE, CD45-PerCP, CD47-Alexa Fluor 647, and CD274-FITC antibodies (all antibodies were obtained from BD Biosciences).
4
Blood Cell Isolation Protocol
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Blood samples were collected EDTA pre-coated 9 mL tubes, and then incubated at 37 °C for 1.5 h. White blood cells were aspirated from thin white layer between plasma and red blood cells after their sedimentation. The obtained cell concentrate washed in 2 mL Cell Wash buffer (BD Biosciences, USA) by centrifugation at 800×g for 15 min.
5
Blood Cell Isolation and Immunophenotyping
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The sample processing and immunophenotyping was performed as described in our previous study11 (link). Blood samples were collected to EDTA pre-coated 9 mL tubes, then incubated at 37 °C for 1.5 h. White blood cells were aspirated from thin white layer between plasma and red blood cells after their sedimentation. Obtained cell concentrate washed in 2 mL Cell Wash buffer (BD Biosciences, USA) by centrifugation at 800× g for 15 min and resuspended in 150 μl of sterile PBS.
6
Multiparametric Flow Cytometry of Cell Markers
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TKTL1, Apo10 and GD2 abundance were detected by flow cytometry. Cells were washed with cell wash buffer (BD Biosciences, Heidelberg, Germany) and were intracellularly stained with the TKTL1 (PE labelled; provided by Zyagnum), Apo10 (FITC labelled; provided by Zyagnum), and GD2 (APC labelled, provided by Prof. Handgretinger) antibodies at room temperature for 20 min. The samples were collected on a CantoII flow cytometer (BD Biosciences) and analysed using FACS Diva v8.0.1 cytometry analysis software (BD Biosciences).
7
Isolation and Characterization of MSCs
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To confirm isolation of MSCs, the cultured cells were collected and immediately incubated with appropriate antibodies for 20 min at room temperature (RT). Then, the cells were washed using Cell Wash Buffer (BD Biosciences) and ultimately suspended in Cell Wash Buffer. Analysis was performed on a BD FACSCanto II flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). Gating parameters dividing positive and negative populations were established based on the signal of isotype IgG control probes. Cells were screened using the Human MSC Analysis Kit (BD Biosciences, no. 562245) for the presence of MSC-associated surface markers (CD73, CD90, and CD105) and the co-occurring absence of blood cell-lineage-specific markers (CD11b, CD19, CD34, CD45, and HLA-DR.29 (link) The phenotype of MSCs infected with vMyx-EGFP (MOI = 10) was also analyzed.
8
Immunophenotyping of Peripheral Blood Cells
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Peripheral blood cells were labelled with fluorochrome-conjugated monoclonal antibodies and resuspended for 20 min at room temperature (RT) in the dark. Red blood cells were lysed by Optilyse C (Beckman Coulter, Brea, CA, USA) for 10 min. The reaction was stopped by CellWash buffer (BD, Franklin Lakes, NJ, USA). The monoclonal antibodies used in the study are summarised in Table 1 .
Samples were measured on the Navios EX flow cytometer (Beckman Coulter, Brea, CA, USA) and analysed using Navios and Kaluza software (Beckman Coulter, Brea, CA, USA). The percentage of positive cells as well as median/mean fluorescence intensity (MFI) were evaluated for each individual marker. Subpopulations of blood monocytes were discriminated as classical (CD14+CD16−), intermediate (CD14+CD16+), and nonclassical (CD14lowCD16+). For the gating strategy, seeFigure 1 .
Samples were measured on the Navios EX flow cytometer (Beckman Coulter, Brea, CA, USA) and analysed using Navios and Kaluza software (Beckman Coulter, Brea, CA, USA). The percentage of positive cells as well as median/mean fluorescence intensity (MFI) were evaluated for each individual marker. Subpopulations of blood monocytes were discriminated as classical (CD14+CD16−), intermediate (CD14+CD16+), and nonclassical (CD14lowCD16+). For the gating strategy, see
9
Phenotypic Characterization of ADSCs
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To confirm the phenotype of ADSCs, cultured cells were incubated with appropriate antibodies (20 min/RT), rinsed with and resuspended in Cell Wash Buffer (BD Biosciences). Analysis was performed using a BD FACS Canto II flow cytometer (Becton-Dickinson). The phenotype of both uninfected and infected ADSCs (vMyx-WT; MOI = 5) was analyzed. The positive cell population gates were set using isotype IgG controls. The presence of ADSC-associated surface markers (CD73, CD90, and CD105) and the co-occurring absence of blood cell-lineage-specific markers (CD11b, CD19, CD34, CD45 and HLA-DR) was examined using the Human MSC Analysis Kit (BD Biosciences, No. 562245).
10
Isolation of White Blood Cells
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Blood samples were collected in EDTA pre-coated 9 mL tubes, then incubated at 37 °C; for 1.5 h. White blood cells were aspirated from a thin white layer between plasma and red blood cells after their sedimentation. Obtained cell concentrate washed in 2 mL Cell Wash buffer (BD Biosciences, San Jose, CA, USA) by centrifugation at 800× g for 15 min.
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