SWHEL mice heterozygous for both light-and heavy-variable
chain alleles of the anti-HEL BCR were sacrificed by cervical dislocation and
splenocytes were collected. Single cell suspensions were obtained by
mechanically disrupting the tissue through 70 μm nylon mesh filters (BD
Biosciences) using complete RPMI 1640 media (Sigma-Aldrich). The exact frequency
of SWHEL B cells was determined by flow cytometry prior to adoptive
transfer using HEL protein conjugated to Alexa Fluor 647 (A647).
SWHEL B cells (CD45.1+) were resuspended in PBS1x and
adoptively transferred (intravenous injection; i.v.) into
congenic C57BL/6 recipient mice (CD45.2+) along with
2×108 sheep red blood cells (SRBCs) conjugated to a
mutated form of hen egg lysozyme (HEL2x or HEL3x) (Paus et al., 2006 (link)). For experiments
analyzing the early stages of the immune response (days 1.0–2.5)
1.5×105 HEL-binding cells were transferred; whereas for
analysis of late phases (days 3–8.5) 3×104 cells were
given.
chain alleles of the anti-HEL BCR were sacrificed by cervical dislocation and
splenocytes were collected. Single cell suspensions were obtained by
mechanically disrupting the tissue through 70 μm nylon mesh filters (BD
Biosciences) using complete RPMI 1640 media (Sigma-Aldrich). The exact frequency
of SWHEL B cells was determined by flow cytometry prior to adoptive
transfer using HEL protein conjugated to Alexa Fluor 647 (A647).
SWHEL B cells (CD45.1+) were resuspended in PBS1x and
adoptively transferred (intravenous injection; i.v.) into
congenic C57BL/6 recipient mice (CD45.2+) along with
2×108 sheep red blood cells (SRBCs) conjugated to a
mutated form of hen egg lysozyme (HEL2x or HEL3x) (Paus et al., 2006 (link)). For experiments
analyzing the early stages of the immune response (days 1.0–2.5)
1.5×105 HEL-binding cells were transferred; whereas for
analysis of late phases (days 3–8.5) 3×104 cells were
given.