Guava easycyte plus

Linear modeling analysis was performed in R version 3.6.0 using the lm function included in the base package⁶⁹ for the 3 CXCL10 traits as follows (see R script in Supplementary Methods): $${\mathit{log}}_2\left(U,n,i,n,f,e,c,t,e,d,\left[C,X,C,L,10\right],\left(p,g,,\ast ,{\mathit{ml}}^{-1}\right)\right)=rs2869462+Population+CellDeath$$ $${\mathit{log}}_2\left(I,n,f,e,c,t,e,d,\left[C,X,C,L,10\right],\left(p,g,,\ast ,{\mathit{ml}}^{-1}\right)\right)=rs2869462+Population+CellDeath+C.trachomatisburden$$ $${\mathit{log}}_2\left(\frac{Infected\left[C,X,C,L,10\right]\left(p,g,,\ast ,{\mathit{ml}}^{-1}\right)}{Uninfected\left[C,X,C,L,10\right]\left(p,g,,\ast ,{\mathit{ml}}^{-1}\right)}\right)=rs2869462+Population+CellDeath+C.trachomatisburden$$ $${\mathit{log}}_2\left(\frac{Infected\left[R,A,N,T,E,S\right]\left(p,g,,\ast ,{\mathit{ml}}^{-1}\right)}{Uninfected\left[R,A,N,T,E,S\right]\left(p,g,,\ast ,{\mathit{ml}}^{-1}\right)}\right)=Population+CellDeath+C.trachomatisburden$$
Variables are modeled in order as written, such that regression for each subsequent variable is calculated on the residual of the preceding variable(s). rs2869462 genotype was modeled as 3 categorical variables: CC, CG, or GG. Population was modeled as a categorical variable with 4 factors: ESN (Esan in Nigeria), GWD (Gambian in Western Divisions in the Gambia), KHV (Kinh in Ho Chi Minh City, Vietnam), and IBS (Iberian in Spain). Cell death was modeled as a continuous variable using the percent of dead cells as measured by 7AAD + staining by flow cytometry. C. trachomatis burden was modeled as a continuous variable as the percent of infected cells measured by flow cytometry of GFP produced by C. trachomatis (LGV-L2 Rif^R pGFP::SW2)-infected LCLs. All flow cytometry traits were measured using a Guava EasyCyte Plus flow cytometer (Millipore).

To assess MSC growth, cells were seeded at 5,000 cells per square centimeter and cultured under maintenance conditions. At 70%–80% confluence, cells were enzymatically removed with 0.125% trypsin/Versene and viable cells counted using Guava EasyCyte Plus (Milipore, Merck KGaA, Darmstadt, Germany, www.merckmillipore.com). Cells were replated at the same density and progressively subcultured for 13–15 passages (i.e., until senescence) to estimate cumulative cell numbers and population doublings. Genomic DNA at different passages was isolated and telomere length analysis performed as described previously 33.

Jurkat, Molt4 and CEM cell lines were generous gifts form the laboratory of Dr. Douglas Graham. Cell lines were tested for mycoplasma and DNA fingerprinted utilizing STR DNA fingerprinting (Life Technologies), as previously described, and stock vials were subsequently stored in liquid nitrogen. Patient derived samples, LAX1 and LAX1R, were obtained at initial diagnosis and relapse, respectively, from a patient with T-ALL after informed consent was obtained, and in compliance with the Institutional Review Board of the University of Southern California. Cells were cultured at 37°C in humidified air supplemented with 5% CO₂ in RPMI supplemented with 10% FBS and antibiotics, except for the LAX1 and LAX1R that were cultured in αMEM with 20% FBS, 2.5% HEPES and antibiotics. Cells were seeded at 2.5 × 10⁵ cells/mL for experimentation and were counted at the indicated time points by propidium iodide (Sigma-Aldrich) exclusion and flow cytometry (Guava EasyCyte Plus, Millipore).

Apoptosis was measured with the Guava EasyCytePlus using Nexin reagent per the manufacturer's protocol (Millipore). Cell cycle, DNA damage and apoptosis were measured simultaneously using the Apoptosis, DNA damage and Cell Proliferation Kit (BD Biosciences) according to the manufacturer's instructions. Fixation and permeabilization buffers from this kit were used to prepare cells for staining with PE-linked human CD45 and phopho-CDK1 antibodies followed by secondary staining with anti-rabbit Alexa Fluor 488. Stained cells were analyzed on a Gallios 561 flow cytometer (Beckman Coulter).