Phytohaemagglutinin-M (PHA-M) is used for the stimulation of cell proliferation in lymphocyte culture. Phytohaemagglutinin (or Phytohemagglutinin) is a lectin extracted from red kidney beans (Phaseolus vulgaris). The protein consists of two molecular species, a leucoagglutinin (PHA-L) and an erythroagglutinin (PHA-E). Each of the proteins contains a family of five isolectins, each being a tetramer held together by noncovalent forces. PHA-M is the mucoprotein form and is a crude extract used for the stimulation of cell proliferation in lymphocyte culture. PHA-M also has a powerful erythroagglutinating property and it was originally used for separating leukocytes from whole blood. Working Concentration: Each ml will contain 5mg of protein
Procedure
1. Add 2-4ml of PHA-M (Cat. No. 12-009-1H) per 100ml Karyotyping Medium (Cat. No. 01-198-1).
2. Inoculate approximately 0.5ml of heparinized whole blood into a glass or plastic tube with 10ml of medium (or 106 viable cells per ml).
3. Incubate the culture for 72 hours.
4. Add 0.1-0.2ml of Colcemid Solution (Cat. No. 12-004- 1) to each culture tube. Incubate the culture for an additional 15-30 minutes.
5. Transfer the culture to a centrifuge tube and spin at 500g for 5 minutes.
6. Remove the supernatant and re-suspend the cells in 5-10ml of hypotonic 0.075M KCl (Cat. No. 12-005-1). Incubate at 37ºC for 10-12 minutes.
7. Spin at 500g for 5 minutes.
8. Remove the supernatant, agitate the cellular sediment and add drop-by-drop 5-10ml of fresh, ice-cold fixative made up of 1 part acetic acid to 3 parts methanol. Leave in 4ºC for 10 minutes.
9. Repeat steps 7 and 8.
10. Spin at 500g for 5 minutes.
11. Re-suspend the cell pellet in a small volume 0.5-1ml of fresh fixative, drop onto a clean slide and allow to air dry.
12. At this stage, the preparation can be stained with Orecin
or Giemsa. Giemsa banding has become the most widely
used technique. The most common method to obtain this
staining is to treat slides with Trypsin-EDTA 10X (Cat.
No. 03-051-5).
- Model 12-009-1H
- Weight: 0.00kg