Lymphocytes were isolated from the spleen and lymph nodes of mice and were prepared into single cell suspensions for flow cytometric analysis and sorting. Lymphocytes were isolated by mashing tissues over 70µm mesh filters. Red blood cells were lysed from cell suspensions with ACK lysis buffer (Sigma) and cells were washed twice with FACs wash prior to FC-block (Biolegend) and antibody staining. Cells were quantified during flow cytometry by the addition of CountBright Absolute Counting beads (Invitrogen) to sample suspensions. For instances where counting beads were unable to be used, quantification was performed by counting with a hemocytometer and percentages from flow cytometry. (NANP)9 tetramers were prepared in house by mixing biotinylated (NANP)9 peptide with streptavidin conjugated PE or APC (Invitrogen) in a 4:1 molar ratio. Live cells were always distinguished with the use of 7-AAD (Biolegend), which was included in the “Dump-gate” channel (which included the markers CD11b and GR1). Flow-cytometric data was collected on a BD Fortessa or X20 flow cytometer (Becton Dickinson) and analyzed using FlowJo software (FlowJo). A BD FACs Aria I or II (Becton Dickinson) machine was used for FACS sorting of cells.
Facs aria 1 or 2
Manufactured by BD
The BD FACs Aria I or II is a cell sorter designed for flow cytometry. It is capable of high-speed cell sorting and analysis. The instrument is equipped with multiple laser options and detection channels to enable flexible and customizable configurations for various applications.
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Lab products found in correlation
3 protocols using facs aria 1 or 2
1
Isolation and Flow Cytometry of Murine Lymphocytes
2
Mouse Lymphocyte Isolation and Flow Cytometry
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Lymphocytes were isolated from the spleen and bone marrow of mice and were prepared into single cell suspensions for flow cytometric analysis and sorting. Bone marrow cells were flushed from femurs and tibias with FACs buffer in 27g syringes, whilst splenocytes were isolated by mashing spleens over 70 mm micron mesh filters. Red blood cells were lysed from cell suspensions with ACK lysis buffer (Sigma) and cells were washed twice with FACs wash prior to Ab staining. Cells were quantified during flow cytometry by the addition of CountBright Absolute Counting beads (Invitrogen) to sample suspensions. Details of Abs are given in Key Resources table, and details of generic gating strategies for mouse experiments are given in Figure S2. (NANP) 9 tetramers were prepared in house by mixing biotinylated (NANP) 9 peptide with streptavidin conjugated PE or APC (Invitrogen) in the a 4:1 molar ratio. Flow-cytometric data was collected on a BD Fortessa or 320 flow cytometer (Becton Dickinson) and analyzed using FlowJo software (FlowJo). A BD FACs Aria I or II (Becton Dickinson) machine was used for FACS sorting of cells.
3
PBMC Sample Preparation for Flow Cytometry
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For samples from Kenya and the IIV cohort PBMCs were thawed and washed in PBS with 2% heat-inactivated FBS. Cells were then stained with Live/Dead dye for 5 min in PBS before incubation with fluorescently labeled antibodies for a further 30 min. Details of all antibodies used are given in the Key resources table . Flow-cytometric data was collected on a BD Fortessa or X20 flow cytometer (Becton Dickinson) and analyzed using FlowJo (FlowJo Software) (). A BD FACs Aria I or II (Becton Dickinson) was used for sorting cells.
For VRC314 clinical trial specimens PBMCs were thawed into prewarmed RPMI media then washed with PBS. Cells were stained with Live/Dead dye for 15 minutes, washed in PBS with 2% heat inactivated FBS, and labeled with antibodies for an additional 30 minutes. Labeled cells were washed with PBS 2% FBS and fixed for 15 minutes in 0.5% PFA before and final wash and resuspension in PBS 2% FBS. Flow-cytometric data was collected on a BD X50 flow cytometer (Becton Dickinson) and analyzed using FlowJo ().
For VRC314 clinical trial specimens PBMCs were thawed into prewarmed RPMI media then washed with PBS. Cells were stained with Live/Dead dye for 15 minutes, washed in PBS with 2% heat inactivated FBS, and labeled with antibodies for an additional 30 minutes. Labeled cells were washed with PBS 2% FBS and fixed for 15 minutes in 0.5% PFA before and final wash and resuspension in PBS 2% FBS. Flow-cytometric data was collected on a BD X50 flow cytometer (Becton Dickinson) and analyzed using FlowJo ().
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