Lifting, Counting Cells & Assays

Question 1

in cell culturing & passaging adherent cells - importance of tissue culture hood (laminar flow hood)?

Answer 1

sterile environment using HEPA-filtered airflow - protects cells from contamination

Question 2

importance of ethanol sterilisation when working with cells?

Answer 2

70% ethanol - optimal for killing microbes (penetrates cells better than 100%)
- fast, non-corrosive, and evaporates - practical gold-standard

Question 3

how does trypsin EDTA help separate & lift cells for detaching?

Answer 3

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

Question 4

why must cells be incubated after trypsin? what must you be cautious about?

Answer 4

allows time for trypsin to digest adhesion proteins - not for too long to avoid damaging cell surface proteins

Question 5

what is DMEM? what does it contain? why is it used as media?

Answer 5

DMEM = Dulbecco’s Modified Eagle Medium - CONTAINS:
- glucose, amino acids, salts, vitamins
- supplemented with FBS (foetal bovine serum) = growth factors
- antibiotics = contamination prevention

Question 6

what is a haemocytometer?

Answer 6

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

Question 7

how is a haemocytometer used to count cells?

Answer 7

• 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

Question 8

formula for cell concentration using a haemocytometer

Answer 8

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

Question 9

how to calculate dilution factor for haemocytometer cell count?

Answer 9

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

Question 10

why is cell counting important?

Answer 10

• ensures consistent seeding of cells across experiments
• measures cell proliferation or death after treatment
• can assess viability using dyes (e.g. Trypan Blue)

Question 11

why spin down cells using a centrifuge after drug treatment?

Answer 11

separates cells from media - removes floating dead cells & leftover drug compounds

allows for a clean cell pellet before viability staining (via trypan blue)

Question 12

why is Accutase sometimes used instead of trypsin?

Answer 12

gentler than trypsin for removing cell adhesion proteins - Accutase preserves surface proteins whilst still detaching adherent cells

Question 13

how does trypan blue measure viability?

Answer 13

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)

Question 14

what is an MTT assay? what does it measure?

Answer 14

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

Question 15

how does an MTT assay work to measure cell metabolic activity/viability?

Answer 15

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)

Question 16

in an MTT assay, treated and untreated control wells are used? why use untreated control wells?

Answer 16

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

Question 17

what does measuring the colorimetric MTT assay at 595nm do?

Answer 17

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

Question 18

what is mechanical cell scrapping? what is it used for?

Answer 18

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

Question 19

pros and cons of mechanical cell scrapping?

Answer 19

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)

Question 20

what is enzymatic scrapping?

Answer 20

using enzymes to break cell adhesion by cleaving surface proteins - e.g. trypsin-EDTA, Accutase

Question 21

what are the two methods of cell scrapping?

Answer 21

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

Question 22

how is trypsin-EDTA used in enzymatic cell scrapping?

Answer 22

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

Question 23

how is Accutase used in enzymatic cell scrapping?

Answer 23

gentler than trypsin - preserves cell surface proteins whilst sill breaking down cell adhesion proteins

Question 24

pros and cons of enzymatic cell scrapping?

Answer 24

pros:
- higher cell viability
- less mechanical damage
- better for reseeding/ downstream assays

cons:
- enzymes may degrade surface proteins (trypsin)
- must neutralise enzyme with serum (e.g., FBS)

Question 25

Q: What is the purpose of using Trypsin-EDTA in cell culture? Give one advantage and one disadvantage. (2 marks)

Answer 25

trypsin-EDTA used for enzymatic cell scrapping in ell culture to separate adherent cells by breaking down cell adhesion proteins advantage: - gentle, efficient detachment; better cell viability for further downstream assays disadvantage: - can damage surface proteins (esp. with trypsin - overexposure); makes it not suitable for experiments where you need intact surface proteins

Question 26

Q: Explain why a researcher might choose mechanical scraping instead of enzymatic lifting when preparing cells for Western blotting. (3 marks)

Answer 26

Western blot - technique that identifies & analyses specific proteins within a complex mixture - enzymatic scrapping may affect/degrade these proteins - especially trypsin - which affects data - mechanical scrapping doesn't degrade proteins; preserves surface proteins better - important to preserve surface proteins for detection in Western Blots

Question 27

Q: You are passaging adherent cells and planning a drug treatment assay. Outline the key steps from detaching the cells to seeding them in a 96-well plate, including why cell counting is important. (5 marks)

Answer 27

- detach cells via a cell scrapping method (enzymatic - trypsin-EDTA/ Accutase; mechanical - physical detaching) - if enzyme scrapping was used = neutralise enzyme with serum-containing medium and collect cells - centrifuge cells before cell counting to remove media & any floating debris - count cells using a haemocytometer & counter & Trypan blue to determine cell viability following drug treatment (live cells = white/clear; dead cells = blue) - calculate required volume to seed a specific number of viable cells per well

Question 28

Q: Compare the use of Trypsin-EDTA and mechanical scraping for lifting adherent cells. In your answer, include when each method is preferred, what reagents are used, and one potential drawback of each. (6 marks)

Answer 28

trypsin-EDTA - enzymatic method of cell scrapping adherent cells from surfaces - digests & breaks down cell adhesion proteins between cell-cell junctions - ADVANTAGE: preserves cell viability better than mechanical scrapping, less physical damage/cell shearing - DISADVANTAGE: 1) enzyme activity may damage/degrade surface proteins - not good if you want to conduct downstream assays (e.g. Western Blots) for detecting surface proteins or phosphorylation. 2) need additional reagents to neutralise enzymes before further assays mechanical scrapping - physically detaching adherent cells using a sterile cell scrapper - ADVANTAGE: 1) doesn't affect surface proteins; 2) don't need additional reagents - DISADVANTAGE: 1) physical damage/cell shearing likely - makes cell viability low for further downstream experiments

Question 29

Q: Describe how a haemocytometer and Trypan Blue are used together to assess both cell number and cell viability. (4 marks)

Answer 29

following treatment of a drug/substance to cell sample - cell suspension and trypan blue mixed in equal parts (1:1 ratio) haemocytometer os a glass slide with a grid etched in it used for manual cell counting - each grid square holds a known volume of fluid = will hold a known volume of cell suspension mixture is added into haemocytometer grid and cell viability is analysed based on trypan blue - live cells have intact cell membranes = trypan blue can't enter = cells are white/clear - dead cells have damaged membranes = trypan blue enters cells = blue cells higher proportion of blue cells = high cell death/ apoptosis following treatment

Question 30

what kind of cells can be used for in vitro study?

Answer 30

non-adherent/suspension cells - adherent cells can be released from the dish by enzymatic or mechanical scrapping

Question 31

how do we harvest cells to make an extract? what method of scrapping is preferred for this and why?

Answer 31

use mechanical scrapping instead of enzymatic - enzymatic can alter the phosphorylation state of some cell surface proteins - can affect data depending on research interest - mechanically scraping cells preserves protein integrity/state - cells are then transferred into a centrifuge & spun to produce a pellet - separates cells from media & contaminants

Question 32

What is the function of Trypsin-EDTA in cell culture? A) To enhance cell adhesion B) To promote cell proliferation C) To detach adherent cells from the culture surface D) To increase cell density

Answer 32

C) To detach adherent cells from the culture surface

Question 33

What is the purpose of Trypan Blue in the cell viability assay? A) To stain dead cells B) To measure cell proliferation C) To enhance cell attachment D) To measure cell size

Answer 33

A) To stain dead cells

Question 34

Which of the following assays directly measures mitochondrial activity as an indicator of cell viability? A) Trypan Blue exclusion B) MTT assay C) Haemocytometer count D) Cell scraping

Answer 34

B) MTT assay