.. _calibration-division: Calibration data for cell cycle and proliferation ------------------------------------------------------------ Assays for calibrating germinal center B cell division """""""""""""""""""""""""""""""""""""""" These are different ways to approach the cell division by using different types of assays. Below are the most used assays for studying cell division. First case -> DNA (preferentially stains dsDNA) Second case -> DNA (measures de novo DNA synthesis or S-phase synthesis of the cell cycle) Third case -> amount of a cytosolic Carboxyfluorescein Succinimidyl ester that is diluted during division Differences in information content """""""""""""""""""""""""""""""""""""""" When using data for calibration these are the things should be take in account: First case gives a snapshot of the DNA distribution does not tell you whether those cells having an S phase DNA content were cycling or noncycling or apoptotic. Second case directly measures de novo DNA synthesis or S-phase synthesis of the cell cycle. Gives the percentage of cell that are in a specific cell cycle phase (S, G2, G1). It is not able to discriminate between one cycle of division or multiple cycles. Plus the BrdU gives information about apoptosis in the form of sub-G1 events Third case gives you the number of all cell cycle division in a cell population for a given time, that goes for days. Those are important differences to take in account when capture data to include in the simulation or to use for validation. DAPI assay from `PMID: 21074050 `_ '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' .. _fig-calibration-dapi: .. figure:: ../img/calibration_dapi.png :figwidth: 90% :align: center Cell division data from DAPI assay. Formal description of DAPI staining '''''''''''''''''''''''''''''''''''''''' **Input**: * Organism: mouse * Cell: Germinal center B cell located in LZ and DZ (photoactivated): * LZ cells phenotype: CXCR4loCD86hiCD83hi * DZ cells phenotype: CXCR4hiCD86loCD83lo * Reagent: Bromodeoxyuridine **Transformation**: * Procedure: Staining * Staining target: DNA ds * Staining reagent: 4',6-diamidino-2-phenylindole (DAPI) * Cell treatment: Fixation and permeabilization * Instrument: FACS **Output**: * Data Type: Facs histograms * Processed data: percentage of cell in different phase of cell cycle CSFE assay From `PMID: 18566403 `_ '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' .. _fig-calibration-cfse: .. figure:: ../img/calibration_cfse.png :figwidth: 90% :align: center Cell division data from CFSE assay. .. _fig-calibration-cfse-comparison: .. figure:: ../img/calibration_cfse_comparison.png :figwidth: 90% :align: center Comparison between the two types of staining on the same sample. Formal description of CFSE staining '''''''''''''''''''''''''''''''''''''''' **Input**: * Organism: mouse * Cell: Naïve B cell * Reagent: Carboxyfluorescein Succinimidyl ester **Transformation** * Procedure: CSFE assay * Target assay: cell cytosol * Reagent: carboxy- fluorescein diacetate, succinimidyl ester * Cell treatment: none * Instrument: FACS **Output**: * Data Type: Facs histograms * Processed data: number of cell divisions Parameters from `PMID: 21074050 `_ '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' **LZ** * DNA synthesis = 14% * DNA in G2/M = 3% * DAPI = 15% **DZ** * DNA synthesis = 20% * DNA in G2/M = 6% * DAPI = 27.6 % Parameters from `PMID: 18566403 `_ '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' **LZ** * DNA Synthesis = 18% * DNA in G2/M = 2.5% * DAPI = 72% **DZ** * DNA Synthesis = 20% * DNA in G2/M = 5% * DAPI = 80% * Number of division of B cell stimulated with anti CD40 and IL-4 for 40 hours * Mean of division = 4 * Each division will take around 10 hours