Pe cy5 mouse anti human cd45 hi30

Peripheral blood samples (4 mL) were prepared in heparinized tubes and incubated with lysis buffer (NH₄, 0.15 M; EDTA, 0.1 mM; KHCO₃, 10 mM; pH = 7.2) at room temperature for 5 min. After centrifugation, the supernatant was discarded, and the cell pellets were washed 2× with PBS. The oHSV1‐hTERT‐GFP virus used in this study, in which the endogenous ICP4 promoter is replaced with the hTERT promoter, has been described in our previous work.²⁶ Following centrifugation, cells were resuspended and transduced with oHSV1‐hTERT‐GFP at a multiplicity of infection of 1 at 37°C in a humidified atmosphere of 5% CO₂ for another 24 h. Thereafter, the cells were collected, and 200 μL of PE‐Cy5 mouse anti‐human CD45 (HI30, BD) was added, followed by incubation in the dark at room temperature for 30 min. After one wash with PBS, the cells were resuspended in 1 mL of PBS. The detection of CD45‐/GFP+ cells from blood samples was performed via flow cytometry (Merck Millipore or BD). CD45‐/GFP+ cells were recorded as positive results.

Peripheral blood samples (4 ml) were prepared in heparinized tubes and incubated with lysis buffer (NH₄Cl, 0.15 M; EDTA, 0.1 mM; KHCO₃, 10 mM; pH = 7.2) at RT for 5 minutes. After centrifugation, supernatant was discarded and cell pellets were washed 2× with PBS. Following centrifugation, cells were resuspended and transduced with oHSV1-hTERT-GFP at an MOI = 1 at 37°C in a humidified atmosphere of 5% CO₂ for another 24 hours. Thereafter, the cells were gathered, 200 μl PE-Cy5 mouse anti-human CD45 (HI30, BD, USA) was added and incubated in the dark at room temperature for 30 minutes. After one wash with PBS, the cells were resuspended in 1 ml PBS. Detection of CD45−/GFP+ cells for solid tumor blood samples were executed by flow cytometry (Merck Millipore, Germany or BD, USA). The CD45−/GFP+ cells were recorded as positive results.