Kaluza analysis version 2

Flow cytometry (Figure 10) is a technique used for measuring the chemical or physical characteristics of cells or particles. The cells or particles of interest are suspended in solution, labelled with fluorescent markers, and run through a cell analyser ‐ a flow cytometer instrument. The cells or particles flow through a small tube one at a time, and a laser is focused on them, scattering light and fluorescence of different wavelengths then recorded by the flow cytometer. Data are presented on a computer as either single parameter histograms or two‐parameter plots called cytograms.
The protocol describing handling and preparation of the blood samples before running on the flow cytometer is described thoroughly in papers II, III and IV (Appendix A page 35).
We used a BD FACSCantoII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and to analyse the data, we used the software Kaluza Analysis (Kaluza Analysis version 2.1; Beckman Coulter, Inc., Pasadena, CA, USA). For measuring white blood cell count (WBC) to obtain 1.0 x 10⁶ leukocytes in the test tubes, we used a Sysmex KX‐21NTM (Sysmex Corporation, Kobe, Japan).
Table 5 is an overview of the monoclonal antibodies used for flow cytometry. A gating strategy for the flow cytometric analyses is shown in paper IV (page 35).

Immunofluorescence flow cytometry was performed as described previously (9 (link)). For mAbs staining, cells were washed with staining buffer (1% FBS in HBSS), resuspended in 50 μl of the same buffer, pre-incubated with purified anti-mouse CD16/32 (Biolegend) for 10 min, and incubated for 30 min at 4°C with each fluorescence-conjugated mAb or isotype control matched with primary antibody. Zombie NIR™ dye (Biolegend) was used to assess live or dead status of cells. The samples were measured using a Gallios flow cytometry or CytoFLEX (Beckman Coulter). Doublets were distinguished from single cells by plotting FSC height vs FCS area. For the isolation of B-ps, fetal liver cells of E15-15.5 mice were immunostained as above, and CD45⁺ LIN^- (CD3^-, CD4^-, CD8^-, CD11b^-, Gr-1^-, NK1.1^-, TER119^-) CD19⁺B220⁺CD93⁺ IgM^-cells were sorted using a Moflo XDP instrument (Beckman Coulter). The purity of the sorted populations constituted more than 95% as determined by a presorted sample run in parallel. Data were analyzed in Kaluza analysis version 2.1 (Beckman Coulter).

The entire mouse femur (bone and medulla) and spleen were suspended in media, and the number of cells were enumerated, washed once with PBS-5% (v/v) FBS, and resuspended in the same solution containing a cocktail of the following antibodies: APC-CD117, APC-Cy7-Sca1, PE-Cy7-CD150, biotin-labeled anti–mouse CD48, and biotin-labeled anti-lineage antibodies. All antibodies were purchased from BD Pharmingen. After 30 minutes of incubation on ice, cells were washed once and stained with streptavidin-PE-Cy5, and cell fluorescence was analyzed with a Gallios analyzer (Beckman Coulter). HPCs were defined as lineage negative (Lin^–) cells. The total hematopoietic stem cell populations were defined as LSK (Lin^–/CD48^neg/c-Kit^pos/Sca-1^pos) and long-term repopulating hematopoietic stem cells were defined by the SLAM phenotype (Lin^–/CD48^neg/c-Kit^pos/Sca-1^pos/CD150^pos) as previously described (79 (link), 80 (link)). Nonspecific signals and dead cells were excluded by appropriate fluorochrome-conjugated isotype and propidium iodide staining, respectively. Results were analyzed with the Kaluza analysis version 2.1 (Beckman Coulter).