Lifting, Counting Cells & Assays Flashcards
in cell culturing & passaging adherent cells - importance of tissue culture hood (laminar flow hood)?
sterile environment using HEPA-filtered airflow - protects cells from contamination
importance of ethanol sterilisation when working with cells?
70% ethanol - optimal for killing microbes (penetrates cells better than 100%)
- fast, non-corrosive, and evaporates - practical gold-standard
when examining cells under a microscope for contamination - what are you looking for?
bacterial/fungal colonies
mycoplasma - invisible without DAPI stain
morphological changes
pH changes - e.g. media colour
how does trypsin EDTA help separate & lift cells for detaching?
trypsin - enzyme that cleaves cell adhesion proteins
EDTA - chelates Ca²⁺/Mg²⁺ ions → weakens cell adhesion
together - detach cells from flask surface by breaking cell adhesion
why must cells be incubated after trypsin? what must you be cautious about?
allows time for trypsin to digest adhesion proteins - not for too long to avoid damaging cell surface proteins
what is DMEM? what does it contain? why is it used as media?
DMEM = Dulbecco’s Modified Eagle Medium - CONTAINS:
- glucose, amino acids, salts, vitamins
- supplemented with FBS (foetal bovine serum) = growth factors
- antibiotics = contamination prevention
what is a haemocytometer?
glass slide etched with a grid - used for manually counting cells under a microscope
holds a known volume of fluid over the grid - allows for calculation of cell concentration
how is a haemocytometer used to count cells?
- load a known volume of cell suspension into the haemocytometer chamber
- chamber has a known depth and grid area, so the volume over each square is known
- count the number of cells in selected squares
- use cell count to calculate cells per ml
formula for cell concentration using a haemocytometer
cell concentration (cells/mL) = (average number of cells per square/ dilution factor) x 10^4
- each square has a volume of 0.0001 mL (10⁻⁴ mL)
- to scale to 1ml = must multiply by 10^4
how to calculate dilution factor for haemocytometer cell count?
dilution factor = tells you how much your original sample was diluted before loading onto the haemocytometer
formula: dilution factor = total volume/ volume of cell suspension/ original sample
e.g. 10ul of cell suspension, 10ul trypan blue - dilution factor is: 10+10/10 = 20/10 = 2 (done a 1:2 dilution; one part cells, 1 part dye)
multiply by DF in the haemocytometer formula to get the original concentration in your cell suspension before dilution
why is cell counting important?
- ensures consistent seeding of cells across experiments
- measures cell proliferation or death after treatment
- can assess viability using dyes (e.g. Trypan Blue)
Gemcitabine, TNF-related apoptosis inducing ligand, chloroquine, and MG132 - what do these all do?
agents are used to induce cell death by different mechanisms—apoptosis, stress, autophagy inhibition (they’re the TREATMENT FOR THE EXPERIMENT)
why spin down cells using a centrifuge after drug treatment?
separates cells from media - removes floating dead cells & leftover drug compounds
allows for a clean cell pellet before viability staining (via trypan blue)
why is Accutase sometimes used instead of trypsin?
gentler than trypsin for removing cell adhesion proteins - Accutase preserves surface proteins whilst still detaching adherent cells
how does trypan blue measure viability?
trypan blue can’t enter intact/live cells but can enter dead cells with damaged membranes following drug treatment
- alive cells = clear/white
- dead cells = blue
more blue cells = more death = more effective treatment (e.g. with testing chemo drugs on cancer cells)
what is an MTT assay? what does it measure?
colorimetric assay - measures cell metabolic activity, which correlates with cell viability (how alive/healthy cells are)
measures viability via mitochondrial reduction of MTT to formazan in live cells
how does an MTT assay work to measure cell metabolic activity/viability?
MTT is a yellow salt
- live cells convert MTT into purple insoluble crystals via mitochondrial enzymes (yellow -> purple)
- dead cells = no metabolism = no colour change (stays yellow)
in an MTT assay, treated and untreated control wells are used? why use untreated control wells?
untreated control wells to compare treatment cells to a baseline measurement - untreated cells should have 100% viability
with MTT - should all convert from yellow to purple
what does measuring the colorimetric MTT assay at 595nm do?
measures how much formazan was made = how many metabolically active cells were present
more formazan/ purple = more live/viable cells that are metabolically active despite treatment
what is mechanical cell scrapping? what is it used for?
what is it:
- when a sterile cell scrapper is used to physically detach adherent cells from the culture surface
when is it used:
- experiments where enzyme exposure may damage membrane proteins, cell enzymes
- protein/ RNA extraction
pros and cons of mechanical cell scrapping?
pros:
- no enzyme = good for preserving surface proteins & sensitive structures
- fast, no incubation time
cons:
- can damage/ shear cells
- low viability for re-seeding
- cell clumping occurs (need to pipette cells up and down to break up clumps)
what is enzymatic scrapping?
using enzymes to break cell adhesion by cleaving surface proteins - e.g. trypsin-EDTA, Accutase
what are the two methods of cell scrapping?
mechanical scrapping - physically detaching adherent cells from surface using a sterilised cell scrapper
enzymatic scrapping - using enzymes to break cell adhesion by cleaving surface proteins
how is trypsin-EDTA used in enzymatic cell scrapping?
trypsin breaks cell adhesion proteins at cell-cell junctions
EDTA chelates Mg2+ and Ca2+ to help detach cells
both weaken cell adhesion proteins & cell-cell junctions = detach cells
