Isolation and Characterization of Splenocytes

Spleens were collected and splenocytes were isolated in a single cell suspension after being passed through a 100 micron filter. Splenocytes were then subjected to red blood cell lysis by treatment with red blood cell lysis buffer (Sigma # R7757) per manufacturer’s recommendation. Peritoneal cells were collected with 10 mLs of cold 1% FBS DMEM. β-lactamase activity was detected using the LiveBLAzer FRET-BG/Loading Kit with CCF2-AM (ThermoFischer Scientific # K1025) as previously described [17 (link),29 (link),32 (link)]. Cell surface antibodies used were CD19-AlexaFluor 700 (clone eBio1D3, eBioscience # 56-0193-81), CD38-APC (clone 90,eBiosciences #17-0382-81), IgD-APC-Cy7 (clone 11-26c.2a, Biolegend # 405716), and CD5-APC (clone 53–7.3, eBioscience # 17-0051-81). Fc blocking antibody 24G2 was used in staining to prevent antibody binding to cellular Fc receptors.

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Niemeyer B.F., Sanford B., Gibson J.E., Berger J.N., Oko L.M., Medina E., Clambey E.T, & van Dyk L.F. (2021). The gammaherpesvirus 68 viral cyclin facilitates expression of LANA. PLoS Pathogens, 17(11), e1010019.

Publication 2021

red blood cell lysis buffer LiveBLAzer FRET-BG/Loading Kit with CCF2-AM IgD-APC-Cy7 (clone 11-26c.2a, CD5-APC (clone 53–7.3,

Antibody Blocking antibody Buffer Cellular Clone Cold Fc receptors Fret Peritoneal Red blood cell Surface antibodies β lactamase

Corresponding Organization : University of Colorado Denver

Other organizations : University of Colorado Anschutz Medical Campus

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Variable analysis

independent variables

Splenocyte isolation method (passing through 100 micron filter)
Red blood cell lysis treatment (using red blood cell lysis buffer)

dependent variables

β-lactamase activity (detected using LiveBLAzer FRET-BG/Loading Kit with CCF2-AM)
Cell surface marker expression (CD19, CD38, IgD, CD5)

control variables

Peritoneal cell collection method (using 10 mL of cold 1% FBS DMEM)
Fc blocking antibody 24G2 for staining

positive controls

Not specified

negative controls

Not specified

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