Anti mac 1

Spleen, lymph nodes, and thymus were isolated during mouse dissection. Bone marrow was isolated from tibias and femurs of mice. Single cell suspension was prepared and red blood cells were depleted by hypotonic lysis. Cells were stained on ice for 30 minutes in PBS containing 3% BSA, 1 mM EDTA, and 0.05% sodium azide with fluorochrome-conjugated antibodies anti-CD3 (BD Biosciences, clone 17A2), anti-CD4 (BD Biosciences, clone GK1.5), anti-CD8 (Caltag, clone CT-CD8a), anti-B220 (Caltag, clone RA3-6B2), anti-IgM (Jackson ImmunoResearch, 115-116-075), anti-Mac1 (BD Biosciences, clone M1/70), anti-Gr1 (eBioscience, clone RB6-8C5). Samples were washed twice with PBS. Data was acquired on LSR II flow cytometric analyzer (BD Biosciences) and analyzed using FlowJo software (Treestar). Total cell numbers were counted on Countess Automated Cell Counter (Invitrogen) and lymphocyte cellularity was determined by multiplying the total cell number of indicated organ by percentage of CD3⁺ or B220⁺ cells within the organ obtained by flow cytometry.

Flow cytometric analysis of the OP9 cells and primary BM cells were performed with the use of FACSCanto (Becton Dickinson, San Jose, CA). The following antibodies were used: anti-PDGFRβ, anti-CD31, anti-CD45, anti-Ter119, anti-CD54 (Biolegend), anti-CD3, anti-CD4, anti-CD8, anti-CD34, anti-Mac-1, anti-Gr-1, anti-B220, and anti-TER-119, anti-NK.1.1, anti-CD106 anti-hCDRA (BD Biosciences), and anti-CD61 anti-CD140a (eBioscience) anti-CD44 anti-hCD45RO (Pharmingen), anti-Notch-1, anti-Notch-4, anti-Jagged-1 (eBioscience), anti-Notch-2, anti-Notch-3, anti-Dll-1, and anti-jagged-2 (Biolegend).

KOP1 or KOBA cells (10⁶ cells) were co-cultured with OP9 cell monolayers in 10-cm dishes in complete BXH2 medium. After 5 days, overgrown KOBA cells were depleted with gentle pipetting from the culture every 2 days. After 8 days, the cultures were treated with trypsin/EDTA and stained with FITC-anti-CD45 antibody, and then CD45⁻ OP9 cells were isolated using FACSAria II (BD Biosciences) or AutoMax (Miltenyl Biotec, Bergisch Gladbach, Germany). In the OP9 cells co-cultured with KOP1 and KOBA cells were called OP9/P and OP9/L, respectively. For in vitro hematopoiesis, B6 BM cells were stained with a mixture of lineage markers (anti-CD45, anti-Ter119 [Biolegend] anti-CD3, anti-CD4, anti-CD8, anti-Mac-1, anti-Gr-1, anti-B220, and anti-NK.1.1 [BD Biosciences]), and the lin⁻ cells were isolated using AutoMax and cultured on OP9 or OP9/L cell monolayers for 5 days. For the Notch ligand assay, C2C12 cell monolayers were cultured in the absence or presence of KOBA cells for 2 days, and the CD45⁻ C2C12 cells were isolated using AutoMax. K562 or MEG-01 cells (10⁶ cells) were co-cultured with OP9 cell monolayers in 10-cm dishes in RPMI medium. After 8 days, the cultures were treated with trypsin/EDTA, stained with human FITC-anti-CD45RA and APC-anti-CD45RO antibody, and CD45⁻ OP9 cells were isolated using AutoMax (Miltenyl Biotec, Bergisch Gladbach, Germany).