lab work 1 &2

Question 1

what is hat media made out of

Answer 1

hypoxanthine
aminopetrin
thymidine

Question 2

what does a positive results mean after the first elisa

Answer 2

the cells in that well from which the supertant came from were hybridomas producing mAbs to CD20

Question 3

how does HAT select for hybridomas

Answer 3

THYMIDINE stops DNA synthesis stopping myleoma cells from proliferating as they have a defett in the HGPRT gene so cannot use the salvage pathway. B cells have te fucntional gene so when there is a fusion the cells surive

Question 4

what make a hybridoma

Answer 4

mice are immunized with extracellular domains of CD20, their B cells are extracred ad fused with immortal myeloma cells

Question 5

what is blocking agent used for

Answer 5

it blocks unoccupied sites of the membrane in ELISA that are sticky

Question 6

how do you get the cells needed to make hybridomas

Answer 6

hemocytes at 10x focus to count the amount
get 1X10 5 so equal amount the centrifudge and decant the supernatant

Question 7

how do you get the cells to fuse

Answer 7

add 1 ml of fusing solution then roatate and then addd 1ml of RPMI and then rotate for 30 seconds then add 10ml over the next minute then centiduge, decant supertanat and add 10ml of HAT media

Question 8

what does a postive result look like for detecting postive fusion

Answer 8

brown dot

Question 9

what do positive dots indicate

Answer 9

the cells in the wells in which the superntant came from were hybridomas producing CD20

Question 10

how is ELISA conducted

Answer 10

2 ul of each supernant addded, add 25ml of blocking solution (milk powder) and incubate while rocking 30 mins
remove milk powder and add 25ml of cnjugate antibody, leave for 30 mins while rockinh
remove conjuagte and add substate, to stop reaction wash the paper

Question 11

what is teh hgrt gene

Answer 11

myleoma cells have a defect in it, normlaly used for generation of nucleosides