Cardset

gin and tonics

find working concentration ranges/ cell line protocols for work with cell line in the specific lab

YOU CANNOT TRUST OTHER GROUP’S WORK

• effect profile (conc, time),

• cytotoxicity/ apoptosis analysis

  …

in later work: what happens if i increase or decrease conc, at what end of conc range am i?

non-differentiated bone marrow stem cells (“blasts”) go into the bloodstream where they proliferate

cancer cells are continuously circulating throughout body, can’t surgically remove

genetic predisposition to failed differentiation; radiation; chemicals (paint thinners); chemotherapy

a) lymphoid (T, B, NK cells)

b) myeloid (erythrocyte, thrombocytes; Neutrophil, Monocyte)

When too high a percentage of blood is unusable proliferating junk, then not enough oxygen is carried around by erythrocytes. Before that it can be entirely unnoticed

fatigue, organ failure, heavy fevers, dysregulated immune system after infection

acute: rapid increase in more undifferentiated cells

chronic: slightly more mature blood cells

ALL / CLL: T and B cell lymphoblasts (adaptive immune system)

AML/ CML: monoblasts (innate immune system)

immortalised CML cells from 29yr old female patient

myeloblasts, Proerythroblast, megakaryoblast (slightly more immature, this is more characteristic for AML according to wiki)

AML cells from 1yr old male patient, monoblasts (more differentiated, only a few steps away from becoming tissue blood cells aka mDCs, macrophages)

DAG analog that activates PKC -> MAP kinase pathway

(has been shown to differentiate THP1 to monocyte)

Active form of vitamin D

Together with vit D receptor it works as a TF for 300-500 genes

polar dye molecule (cannot pass membrane, there are no transporters)

-> PI only stains cells with porous membranes aka dying cells

-> PI intercalates with DNA when inside cell

1. narrow capillary with 1 cell at a time

2. laser goes through to forward scatter detector (FSC) to determine cell size

3. laser is scattered depending on complexity of cell shape/ granularity (side detector : SSC)

4. cells sorted according to a charge/ surface marked thingy

Plot FSC/ SSC to get points that show groups of cell types in sample

Cytotoxicity/ cell viability %

distribution of fluorescent signal:

• “dark” - live cells (own fluorescence)

• “bright” - dying cells

Positive: lysis buffer added to non treated cells

Negative: non treated sample without a staining to get the own-fluorescence of cells

(5% of cells are always dead btw)

What we did:

• kill cells with acid

• Wash away suspension and all

• Add SRB dye under acidic conditions (SRB binds to basic amino acids)

• wash excess and change pH back to slightly basic -> dye is now in solution

• Measure conc in each well with plate reader

This tells us how much cell mass was adherent! (aka differentiated?)

all are colorimetric assays of viability

SRB can also be used for cell viability analysis

LDH: endogenous to cells reduces NAD to NADH; we test how much of it is outside cells by luciferin

MTT: cellular metabolic activity measured: mitochondiral enzyme activity